Chemical compounds

ABSTRACT

Compounds of formula (I)  
                 
 
in which R 1 , R 2  and X 4  have the meanings given in the specification are Factor Xa inhibitors useful in the treatment of thrombotic disorders.

This application claims the benefit of U.S. Provisional Applications No.60/307,634, filed Jul. 26, 2001; 60/311,462, filed Aug. 13, 2001; and60/339,317, filed Dec. 12, 2001, each of which is incorporated byreference herein.

The present invention relates to compounds useful as pharmaceuticals, topharmaceutical compositions comprising the compounds, to a process forpreparing the compounds, to intermediates useful in the preparation ofthe compounds, and to use of the compounds as pharmaceuticals.

Cardiovascular disease continues to present a major worldwide healthproblem, and is a common cause of serious illness and death.

One line of investigation being pursued by researchers in the search fornew treatments for cardiovascular disease is based upon the hypothesisthat an inhibitor of the serine protease, Factor Xa, may be useful as ananticoagulant agent in the treatment of thrombotic disease.

Inhibitors of Factor Xa are known. For example, WO 99/11657, WO 99/11658and WO 00/76971 disclose certain compounds containing an aromatic group,a glycine residue that bears a cyclic group and a lipophilic group. WO99/11657, which discloses compounds in which the aromatic group is anaminoisoquinoline group, also generically discloses aminoisoquinolinecompounds containing a glycine residue that bears an acyclic group.

Surprisingly, compounds containing particular phenyl or indolyl groups,a glycine residue bearing an alkyl group and a4-(1-methylpiperidin-4-yl)piperidin-1-yl or4-(1-methyl-piperidin-4-yl)piperazin-1-yl group have now been found thatare selective Factor Xa inhibitors and have particularly advantageousproperties.

Accordingly, the present invention provides a compound of formula (I)

in which

-   -   R¹ represents (1-4C)alkyl, (2-4C)alkenyl or (2-4C)alkynyl; and    -   R² is selected from        in which    -   X¹ represents a hydrogen atom or a halogen atom;    -   X² represents a hydrogen atom, a methyl group, a chlorine atom        or a bromine atom;    -   X³ represents a hydrogen atom, a methyl group or a halogen atom;    -   X⁵ represents chloro, methoxy or methyl;    -   X⁶ represents a hydrogen atom, a halogen atom or a methyl group;        and

X⁴ represents CH or N;

or a pharmaceutically acceptable salt thereof.

Compounds of formula (I) have been found to be potent and selectiveinhibitors of the serine protease, Factor Xa, to have good anticoagulantactivity in human plasma, to have good plasma exposure upon oraladministration to mammals, and to possess particularly advantageouspharmacological and toxicological profiles of activity.

In one group of compounds of formula (I), the group R² is selected from

In another group of compounds of formula (I), the group R² is selectedfrom

In yet another group of compounds of formula (I), the group R² isselected from

R¹ is preferably a (1-4C)alkyl group.

Examples of particular values for R¹ are methyl, ethyl, propyl,2-propyl, butyl, t-butyl, 1-methylpropyl and 2-methylpropyl. Furtherexamples are prop-3-enyl and prop-3-ynyl. When R¹ is 1-methylpropyl, thepreferred stereochemistry is that corresponding to (D)-isoleucine.

Preferably, R¹ is 2-propyl.

X¹ preferably represents a hydrogen atom or a fluorine atom.

X² preferably represents a hydrogen atom or a chlorine atom.

X³ preferably represents a fluorine or chlorine atom, such as a chlorineatom.

X⁶ preferably represents a chlorine atom.

Examples of particular values for R² are 4-methoxyphenyl, indol-6-yl,3-methylindol-6-yl, 3-chloroindol-6-yl and 5-chloroindol-2-yl. Furtherexamples are 3-fluoro-4-methoxyphenyl, 5-fluoroindol-2-yl and6-chlorobenzo[b]-thiophen-2-yl. Particular mention may be made ofcompounds of formula (I) in which R² is 3-fluoro-4-methoxyphenyl and ofcompounds of formula (I) in which R² is 5-fluoroindol-2-yl.

R² is preferably 4-methoxyphenyl, indol-6-yl or 5-chloroindol-2-yl.

One particular value for X⁴ is CH.

When X⁴ represents CH, especially preferred compounds of formula (I)are:—

-   -   1-(4-methoxybenzoyl-D-valinyl)-4-(1-methylpiperidin-4-yl)-piperidine        and    -   1-(indole-6-carbonyl-D-valinyl)-4-(1-methylpiperidin-4-yl)piperidine;    -   and pharmaceutically acceptable salts thereof, particularly the        methanesulfonic acid salt of the former.

Another particular value for X⁴ is N.

When X⁴ represents N, an especially preferred compound of formula (I)is:—

-   -   1-(5-chloroindole-2-carbonyl-D-valinyl)-4-(1-methyl-piperidin-4-yl)piperazine,    -   and pharmaceutically acceptable salts thereof.

Examples of compounds of formula (I) in which R² represents

are:

-   1-(3-amino-4-chlorobenzoyl-D-valinyl)-4-(1-methylpiperidin-4-yl)piperidine;-   1-(3-amino-4-chlorobenzoyl-D-valinyl)-4-(1-methylpiperidin-4-yl)piperazine;-   1-(3-amino-4-methoxybenzoyl-D-valinyl)-4-(1-methylpiperidin-4-yl)piperidine;-   1-(3-amino-4-methoxybenzoyl-D-valinyl)-4-(1-methylpiperidin-4-yl)piperazine;-   1-(3-amino-4-methylbenzoyl-D-valinyl)-4-(1-methylpiperidin-4-yl)piperidine;-   1-(3-amino-4-methylbenzoyl-D-valinyl)-4-(1-methylpiperidin-4-yl)piperazine;-   and pharmaceutically acceptable salts thereof.

It will be appreciated that the compounds of formula (I) contain acenter of asymmetry that has the (D) configuration. This is theconformation that would result from construction from a D-α-amino acidH₂N—CH(R¹)COOH. The compounds may therefore exist and be isolated in amixture with the corresponding (L) isomer, such as a racemic mixture, orseparately. Preferably the compounds are isolated substantially free ofthe (L) isomer.

It will also be appreciated that the compounds of formula (I) or theirpharmaceutically acceptable salts may be isolated in the form of asolvate, and accordingly that any such solvate is included within thescope of the present invention.

In the compounds according to the invention, unless otherwise indicated,examples of the term “halogen atom” are fluoro and chloro. The specificname of an alkyl group, such as propyl or butyl, signifies theunbranched or n-isomer, unless otherwise indicated.

The compounds of formula (I) and their pharmaceutically acceptable saltsmay be prepared by a process, which comprises:

-   -   (a) reacting a compound of formula (II)        or a salt thereof, with a compound of formula (III)        or a reactive derivative thereof; or    -   (b) reacting a compound of formula (IV)        or a salt thereof, with a compound of formula (V)        HOOC—R²  (V)        or a reactive derivative thereof;    -   followed, if a pharmaceutically acceptable salt is desired, by        forming a pharmaceutically acceptable salt.

The reaction between a compound of formula (II) with a compound offormula (III) may conveniently be performed employing reagents andreaction conditions conventionally used for the formation of an amidebond. The reaction is conveniently carried out in the presence of abenzotriazole-based reagent such as 1-hydroxybenzotriazole or1-hydroxy-7-azabenzotriazole and a dehydrating agent such asdicyclohexyl-carbodiimide or1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, in an inert organicsolvent such as dimethylformamide and/or methylene chloride. Thereaction is conveniently conducted at a temperature of from 0 to 50° C.,preferably at ambient temperature. If a salt of a compound of formula(II) is used, the reaction is conveniently performed in the additionalpresence of a base such as triethylamine. Other suitable reagents andsolvents are known in the art, for example an acid halide, such as thechloride in the presence of a base, such as triethylamine.

The reaction between a compound of formula (IV) with a compound offormula (V) may conveniently be performed employing reagents andreaction conditions conventionally used for the formation of an amidebond. The reaction is conveniently carried out in the presence of abenzotriazole-based reagent such as 1-hydroxybenzotriazole or1-hydroxy-7-azabenzotriazole and a dehydrating agent such asdicyclohexylcarbodiimide or1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, in an inert organicsolvent such as dimethylformamide and/or methylene chloride. Thereaction is conveniently conducted at a temperature of from 0 to 50° C.,preferably at ambient temperature. If a salt of a compound of formula(IV) is used, the reaction is conveniently performed in the additionalpresence of a base such as triethylamine. Other suitable reagents andsolvents are known in the art, for example an acid halide, such asp-anisoyl chloride in the presence of a base, such as triethylamine.Alternatively, the compound of formula (IV) may be reacted with acompound of formula (V) in the presence of diethyl cyanophosphonate.This reaction is conveniently performed in an organic solvent such asdichloromethane in the presence of a base, such as triethylamine. Thetemperature is conveniently in the range of from −25 to 25° C.

The compound of formula (II) in which X⁴ is CH is known, for examplefrom WO 00/76971 at pages 163-164, and is named as4-(1-methylpiperidin-4-yl)piperidine or 1-methyl-4,4′-bispiperidine. Thecompound of formula (II) in which X⁴ is N is commercially available. Itis referred to herein as 4-(1-methylpiperidin-4-yl)piperazine, but alsomay be named 1-(1-methylpiperidin-4-yl)piperazine.

The compounds of formula (III) may be prepared by reacting a compound offormula (VI)

in which R³ represents a carboxyl protecting group, for example a(1-6C)alkyl group, such as methyl or ethyl, with a compound of formula(IV) to afford a compound of formula (VII)

followed by removing the protecting group.

The compounds of formula (IV) may be prepared by reacting a compound offormula (II) with a compound of formula (VIII)

in which R⁴ represents an amino protecting group, such ast-butoxycarbonyl (Boc) to afford a compound of formula (IX)

followed by removing the protecting group.

The compounds of formulae (VI) and (VIII) are known or may be preparedusing conventional methods for the preparation of amino acids protectedon the carboxy or amino group.

The compounds of formula (V) are well known.

The protection of amino and carboxylic acid groups is described inMcOmie, Protecting Groups in Organic Chemistry, Plenum Press, NY, 1973,and Greene and Wuts, Protecting Groups in Organic Synthesis, 2nd. Ed.,John Wiley & Sons, NY, 1991. Examples of carboxy protecting groupsinclude C₁-C₆ alkyl groups such as methyl, ethyl, t-butyl and t-amyl;aryl(C₁-C₄)alkyl groups such as benzyl, 4-nitrobenzyl, 4-methoxybenzyl,3,4-dimethoxybenzyl, 2,4-dimethoxybenzyl, 2,4,6-trimethoxybenzyl,2,4,6-trimethylbenzyl, benzhydryl and trityl; silyl groups such astrimethylsilyl and t-butyldimethylsilyl; and allyl groups such as allyland 1-(trimethylsilylmethyl)prop-1-en-3-yl.

Examples of amine protecting groups include acyl groups, such as groupsof formula RCO in which R represents C₁₋₆ alkoxy, phenyl C₁₋₆ alkoxy, ora C₃₋₁₀ cycloalkoxy, wherein a phenyl group may be optionallysubstituted, for example by one or two of halogen, C₁-C₄ alkyl and C₁-C₄alkoxy.

Preferred amino protecting groups include benzyloxycarbonyl (Cbz) andt-butoxycarbonyl (Boc).

Certain of the intermediates described herein, for example the compoundsof formulae (III) and (IV), are believed to be novel and accordingly areprovided as further aspects of the invention.

The compounds of the invention may be administered by any convenientroute, e.g. into the gastrointestinal tract (e.g. rectally or orally),the nose, lungs, musculature or vasculature or transdermally. Thecompounds may be administered in any convenient administrative form,e.g. tablets, powders, capsules, solutions, dispersions, suspensions,syrups, sprays, suppositories, gels, emulsions, patches etc. Suchcompositions may contain components conventional in pharmaceuticalpreparations, e.g. diluents, carriers, pH modifiers, sweeteners, bulkingagents, and further active agents. If parenteral administration isdesired, the compositions will be sterile and in a solution orsuspension form suitable for injection or infusion. Such compositionsform a further aspect of the invention.

Viewed from this aspect the invention provides a pharmaceuticalcomposition, which comprises the compound of formula (I) or apharmaceutically acceptable salt thereof, together with apharmaceutically acceptable diluent or carrier.

According to another aspect, the present invention provides a compoundof formula (I) or a pharmaceutically acceptable salt thereof, for use intherapy.

According to another aspect, the present invention provides the use of acompound of formula (I) or a pharmaceutically acceptable salt thereof,for the manufacture of a medicament for the treatment of a thromboticdisorder.

According to another aspect, the present invention provides a method oftreating a thrombotic disorder in a subject requiring treatment, whichcomprises administering an effective amount of a compound of formula (I)or a pharmaceutically acceptable salt thereof.

The subject may be a human or a non-human animal, such as a non-humanmammal, for example a cat, dog, horse, cow or sheep.

The thrombotic disorder may be, for example, venous thrombosis,pulmonary embolism, arterial thrombosis, myocardial ischaemia,myocardial infarction or cerebral thrombosis. A particular indicationis, for example, prophylaxis of post-operative venous thrombosisfollowing high risk orthopedic surgery (such as hip or kneereplacement), primary treatment of venous thrombosis, secondaryprevention of ischemic cardiovascular complications following myocardialinfarction (in combination with e.g. low dose aspirin), or prevention ofembolic stroke in non-valvular atrial fibrillation. The compounds mayalso be used in accordance with the method of the invention in thetreatment of acute vessel closure associated with thrombolytic therapyand restenosis, for example after transluminal coronary angioplasty orbypass grafting of the coronary or peripheral arteries, and in themaintenance of vascular access patency in long term hemodialysispatients.

The dosage of the compound of formula (I) will depend upon the natureand severity of the condition being treated, the administration routeand the size and species of the subject. In general, quantities in therange of from 0.01 to 100 μM/kg bodyweight will be administered.

As used herein, the term “treatment” includes prophylactic use. The term“effective amount” refers to the amount of the compound of formula (I)that is effective to reduce or inhibit the development of the symptomsof the thrombotic disorder being treated.

The compound according to the invention may be administered alone or incombination with an anticoagulant having a different mode of action orwith a thrombolytic agent.

The following Examples illustrate the invention.

Abbreviations used follow IUPAC-IUB nomenclature. Additionalabbreviations are Boc, tertiary-butyloxycarbonyl; DCC,dicyclohexylcarbodiimide; DIEA, N,N-diisopropylethylamine; DMSO,dimethyl sulfoxide (perdeuterated if for NMR); DMF, dimethylformamide;EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;ES-MS, electrospray mass spectrum; EtOAc, ethyl acetate; Et₂O, diethylether; HOAt, 1-hydroxy-7-azabenzotriazole; HOBt, 1-hydroxybenzotriazole; HPLC, high pressure liquid chromatography; MeOH,methanol; SCX, strong cation exchange; TEA, triethylamine; TFA,trifluoroacetic acid; and THF, tetrahydrofuran. Reagents were obtainedfrom a variety of commercial sources.

General Coupling Methods

Method 1: A solution or suspension of an amine or amine hydrochloridesalt (1 molar equivalent, approximately 0.2 M) in THF, dichloromethane,or DMF (or a mixture of any of these solvents) is treated with acarboxylic acid (approximately 1 molar equivalent), either HOBt or HOAt(approximately 1 molar equivalent), either TEA or DIEA (0-3 molarequivalent), and either EDCI or DCC (approximately 1 molar equivalent).After stirring overnight at room temperature, the solvents are removedand the residue is diluted with ethyl acetate or dichloromethane andwashed with saturated aqueous sodium bicarbonate. The organic solutionis then dried with MgSO₄, filtered and concentrated in vacuo. Ifnecessary, the product is purified by chromatography over silica gel,eluting with a gradient of 0% through 2 to 12% (2 N ammonia/methanol) indichloromethane or chloroform. The product-containing fractions are thencombined and concentrated in vacuo.

Method 2: To a stirring solution of an amine or amine hydrochloride salt(1 molar equivalent), triethylamine (1-3 molar equivalent), and acarboxylic acid (about 1.2 molar equivalent) in dichloromethane (0.2-0.5M) at 0° C., is slowly added diethyl cyanophosphonate (about 1.2 molarequivalent). After stirring overnight, the solvents are removed invacuo; and the residue is partitioned between water and an organicsolvent, such as ethyl acetate or dichloromethane, and washed withsaturated aqueous NaHCO₃, followed by brine. The organic phase is thendried with MgSO₄ or Na₂SO₄, filtered and concentrated in vacuo. Ifnecessary, the product is purified by chromatography over silica gel,eluting with a gradient of 0-10% (2 N ammonia/methanol) in eitherdichloromethane or chloroform. The product-containing fractions are thencombined and concentrated in vacuo.

Method 3: The amine or amine hydrochloride salt (1 molar equivalent) andtriethylamine (1-3 molar equivalent) are dissolved in dichloromethane(0.2-0.5 M) and an acid chloride (about 1.2 molar equivalent) is added.After stirring for about 3 h, the volatiles are removed in vacuo; andthe residue is dissolved in methanol (possibly with an organic cosolventsuch as dichloromethane) and loaded onto a strong cation exchange (SCX)column. The column is washed with methanol, and then the desired productis eluted from the column with a solution of ammonia or triethylamine inmethanol (possibly with an organic cosolvent such as dichloromethane).The product containing fractions are then combined and concentrated invacuo. If necessary, the product can be purified further bychromatography over silica gel, eluting with a gradient of 0-10% (2 Nammonia/methanol) in either dichloromethane or chloroform. Theproduct-containing fractions are then combined and concentrated invacuo.

General Deprotection Methods

Method 1: A solution of the t-butyl carbamate (1 molar equivalent) inCH₂Cl₂ (0.2 M) is treated with anisole (5 eq) and TFA (20% by volume).After stirring 1 to 3 h at ambient temperature, the reaction mixture isconcentrated in vacuo. The crude residue (TFA salt) is purified bystrong cation exchange chromatography (SCX). The SCX column is washedwith a 5% solution of acetic acid in methanol, and the TFA salt isdissolved in methanol (possibly with a cosolvent such asdichloromethane) and loaded onto the SCX column. The column is thenwashed with methanol (possibly with a cosolvent such as dichloromethane)and then the free base is eluted from the column with a 2 N solution ofammonia or triethylamine in methanol (possibly with a cosolvent such asdichloromethane). The product containing fractions are then combined andconcentrated in vacuo to give the product in the free base form.

Method 2: HCl gas is bubbled into a solution of the t-butylcarbamate inanhydrous MeOH (0.1 M) for approximately 10 to 30 min; then the reactionmixture is concentrated in vacuo to give a hydrochloride salt of thetitle amine.

General HCl Salt Formation Methods

Method 1: The free base is dissolved in 0.2 N aqueous HCl (1-2equivalents of HCl). The resulting solution is freeze-dried to give theamine hydrochloride salt.

Method 2: A solution of the free base in a small amount of CH₂Cl₂ istreated with 1.0-2.2 equivalents of 1 M HCl in ether. After stirring 30min, the reaction mixture is filtered, and the resulting solid is rinsedwith ether and dried to give the amine hydrochloride salt.

General Analytical HPLC Methods

Method 1: Vydac C18 (4.6×250 mm column), elute with a linear gradient of90/10 through 50/50 (0.1% TFA in water/0.1% TFA in acetonitrile) over 40min, 1 mL/min, λ=214 nm.

Method 2: Xterra RP18 (4.6×50 mm column), gradient of 2-50% CH₃CN in H₂Owith 0.1% TFA, 1 mL/min, over 30 min, λ=214 nm.

Method 3: Xterra RP18 (4.6×150 mm column), gradient of 10-50% CH₃CN inH₂O with 0.1% TFA, 1 mL/min, over 40 min, Waters 996 PDA and/or SedexELS detection.

Preparation of Intermediates

1-(Boc-D-alaninyl)-4-(1-methylpiperidin-4-yl)piperidine.

Prepared from Boc-D-alanine and 4-(1-methylpiperidin-4-yl)piperidinedihydrobromide using methods substantially equivalent to GeneralCoupling Method 1.

¹H NMR.

ES-MS, m/z 354.3 (M+1)⁺.

Analysis for C₁₉H₃₅N₃O₃ 0.3H₂O: Calcd: C, 63.58; H, 10.00; N, 11.71;Found: C, 63.46; H, 9.82; N, 11.61.

1-(Boc-D-ethylglycinyl)-4-(1-methylpiperidin-4-yl)piperidine.

Prepared from Boc-D-ethylglycine and4-(1-methylpiperidin-4-yl)piperidine dihydrobromide using methodssubstantially equivalent to General Coupling Method 1.

¹H NMR.

ES-MS, m/z 368.3 (M+1)⁺.

Analysis for C₂₀H₃₇N₃O₃0.2H₂O: Calcd: C, 64.72; H, 10.16; N, 11.32;Found: C, 65.03; H, 9.98; N, 10.94.

1-[Boc-D-(propyl)glycinyl]-4-(1-methylpiperidin-4-yl)-piperidine.

Prepared from Boc-D-(propyl)glycine and4-(1-methyl-piperidin-4-yl)piperidine dihydrobromide using methodssubstantially equivalent to General Coupling Method 1.

¹H NMR.

ES-MS, m/z 382.3(M+1)⁺.

Analysis for C₂₁H₃₉N₃O₃1.3H₂O: Calcd: C 62.28; H 10.35; N 10.38; Found:C 62.29; H 9.77; N 10.01.

1-[Boc-D-(butyl)glycinyl]-4-(1-methylpiperidin-4-yl)piperidine.

Prepared from Boc-D-(n-butyl)glycine and4-(1-methyl-piperidin-4-yl)piperidine dihydrobromide using methodssubstantially equivalent to General Coupling Method 1.

¹H NMR.

ES-MS, m/z 396.3(M+1)⁺.

Analysis for C₂₂H₄₁N₃O₃: Calcd: C, 66.80; H, 10.45; N, 10.62; Found: C,66.68; H, 10.41; N, 10.50.

1-(Boc-D-Valinyl)-4-(1-methylpiperidin-4-yl)piperidine.

To a solution of Boc-D-Val-OH (33.0 g, 152 mmol) in anhydrous DMF (1.5L) is added 4-(1-methylpiperidin-4-yl)-piperidine dihydrobromide (52.2g, 152 mmol) [4-(1-methyl-piperidin-4-yl)piperidine dihydrobromide isalso named 1-methyl-4,4′-bispiperidine dihydrobromide; and itspreparation is described in WO 00/76971 at pages 163-164.] followed bytriethylamine (63.5 mL, 456 mmol), HOBt (20.5 g, 152 mmol) and DCC (31.3g, 152 mmol) at room temperature. The reaction is allowed to stir atroom temperature overnight. The mixture is then poured into aqueous LiCl(1.5 L) and extracted with CH₂Cl₂ (3×2 L). The organic layers arecombined, dried over Na₂SO₄, filtered and concentrated in vacuo. Theresidue is dissolved in CHCl₃ (1 L), filtered and concentrated in vacuo.The crude material is purified using chromatography over silica gel,eluting with 4% (2 M NH₃ in MeOH)/CHCl₃ to give 43.3 g (75%) of thetitle compound as a tan foam.

¹H NMR (DMSO-d₆) δ 6.68 (m, 1H), 4.42 (m, 1H), 4.19 (m, 1H), 2.94 (m,1H), 2.76 (m, 2H), 2.47 (m, 1H), 2.11 (s, 3H), 1.89 (bs, 1H), 1.8-1.5(bm, 6H), 1.37 (s, 9H), 1.2-0.9 (bm, 7H), 0.82 (m, 6H).

ES-MS, m/z 382.3 (M+1)⁺.

1-(Boc-D-leucinyl)-4-(1-methylpiperidin-4-yl)piperidine.

Prepared from Boc-D-leucine and 4-(1-methylpiperidin-4-yl)piperidinedihydrobromide using methods substantially equivalent to GeneralCoupling Method 1.

ES-MS, m/z 396.4(M+1)⁺.

1-[Boc-D-(t-butyl)glycinyl]-4-(1-methylpiperidin-4-yl)-piperidine.

Prepared from Boc-D-(t-butyl)glycine and4-(1-methyl-piperidin-4-yl)piperidine dihydrobromide using methodssubstantially equivalent to General Coupling Method 1.

¹H NMR.

ES-MS, m/z 396.3(M+1)⁺.

Analysis for C₂₂H₄₁N₃O₃1.3H₂O: Calcd: C, 64.73; H, 10.47; N, 10.29;Found: C, 64.65; H, 10.03; N, 10.30.

1-(Boc-D-valinyl)-4-(1-methylpiperidin-4-yl)piperazine.

To a stirring solution of Boc-D-valine (5.0 g, 23 mmol) indichloromethane (230 mL) is added HOBt (3.42 g, 25.3 mmol), followed byEDCI (4.85 g, 25.3 mmol), followed by4-(1-methylpiperidin-4-yl)piperazine (4.2 g, 23 mmol). After stirringovernight, the solution is washed with saturated aqueous NaHCO₃,followed by brine, and then concentrated in vacuo. The residue ischromatographed over silica gel, eluting with a gradient of 0-10% (2 Nammonia/methanol) in chloroform. The product-containing fractions arecombined and concentrated in vacuo to give the title product (8.98 g,quantitative) as an off-white solid.

¹H NMR.

ES-MS, m/z 383.3(M+1)⁺.

Analysis for C₂₂H₄₁N₃O₃·0.5H₂O: Calcd: C, 61.35; H, 10.04; N, 14.31;Found: C, 61.50; H, 9.68; N, 14.29.

1-[Boc-D-(t-butyl)glycinyl]-4-(1-methylpiperidin-4-yl)-piperazine.

Prepared from Boc-D-(t-butyl)glycine and4-(1-methyl-piperidin-4-yl)piperazine using methods substantiallyequivalent to General Coupling Method 1.

¹H NMR.

ES-MS, m/z 397.3(M+1)⁺.

Analysis for C₂₁H₄₀N₄O₃0.5H₂O: Calcd: C, 62.19; H, 10.1; N, 13.81;Found: C, 62.08; H, 9.89; N, 13.88.

1-(Boc-D-isoleucinyl)-4-(1-methylpiperidin-4-yl)piperazine.

Prepared from Boc-D-isoleucine and 4-(1-methyl-piperidin-4-yl)piperazineusing methods substantially equivalent to General Coupling Method 1.

¹H NMR.

ES-MS, m/z 397.4 (M+1)⁺.

Analysis for C₂₁H₄₀N₄O₃0.2H₂O-0.2CH₂Cl₂: Calcd: C, 61.04; H, 9.86; N,13.43; Found: C, 60.87; H, 9.53; N, 13.60.

1-(Boc-D-leucinyl)-4-(1-methylpiperidin-4-yl)piperazine.

Prepared from Boc-D-leucine and 4-(1-methylpiperidin-4-yl)piperazineusing methods substantially equivalent to General Coupling Method 1.

ES-MS, m/z 397.3 (M+1)⁺.

1-D-Alaninyl-4-(1-methylpiperidin-4-yl)piperidine.

Prepared from 1-(Boc-D-alaninyl)-4-(1-methylpiperidin-4-yl)piperidineusing methods substantially equivalent to those described in GeneralDeprotection Method 1.

¹H NMR.

ES-MS, m/z 254.2 (M+1)⁺.

1-D-Ethylglycinyl-4-(1-methylpiperidin-4-yl)piperidine hydrochloride.

Prepared from1-(Boc-D-ethylglycinyl)-4-(1-methyl-piperidin-4-yl)piperidine usingmethods substantially equivalent to those described in GeneralDeprotection Method 2.

¹H NMR.

ES-MS, m/z 268.2 (M+1)⁺.

Analysis for C₁₅H₂₉N₃O·2.7 HCl·0.6H₂O: Calcd: C, 47.60; H, 8.82; N,11.10; Found: C, 47.59; H, 8.82; N, 10.97.

1-D-(Propyl)glycinyl-4-(1-methylpiperidin-4-yl)piperidine hydrochloride.

Prepared from1-[Boc-D-(propyl)glycinyl]-4-(1-methyl-piperidin-4-yl)piperidine usingmethods substantially equivalent to those described in GeneralDeprotection Method 2.

¹H NMR.

ES-MS, m/z 282.3 (M+1)⁺.

Analysis for C₁₆H₃₁N₃O·2.6 HCl·1.6H₂O: Calcd: C, 47.44; H, 9.16; N,10.37; Found: C, 47.35; H, 9.18; N, 10.29.

1-D-(Butyl)glycinyl-4-(1-methylpiperidin-4-yl)piperidine hydrochloride.

Prepared from1-[Boc-D-(butyl)glycinyl-4-(1-methyl-piperidin-4-yl)piperidine usingmethods substantially equivalent to those described in GeneralDeprotection Method 2.

¹H NMR.

ES-MS, m/z 296.3 (M+1)⁺.

Analysis for C₁₇H₃₃N₃O·2.6 HCl1.6H₂O: Calcd: C, 51.61; H, 9.30; N,10.62; Found: C, 51.59; H, 9.27; N, 10.51.

1-D-Valinyl-4-(1-methylpiperidin-4-yl)piperidine Dihydrochloride.

1-(Boc-D-Valinyl)-4-(1-methylpiperidin-4-yl)piperidine (23.3 g, 61.1mmol) is dissolved in anhydrous methanol (700 mL), cooled to 0° C. andtreated with HCl (gas) until HPLC indicates consumption of startingmaterial is complete. The solvent is removed in vacuo to give 20.8 g(96%) of the title compound as an off-white foam.

¹H NMR (DMSO-d₆) δ 11.8 (bs, 1H), 8.19 (m, 3H), 4.77 (bs, 3H), 4.44 (m,1H), 4.22 (m, 1H), 3.99 (m, 1H), 3.1-2.6 (bm, 6H), 2.01 (m, 1H), 1.9-1.5(bm, 5H), 1.3 (m, 2H), 0.98 (m, 3H), 0.91 (m, 3H).

ES-MS, m/z 282.3 (M+1)⁺.

1-D-Leucinyl-4-(1-methylpiperidin-4-yl)piperidine.

Prepared from 1-(Boc-D-leucinyl)-4-(1-methylpiperidin-4-yl)piperidineusing methods substantially equivalent to those described in GeneralDeprotection Method 1.

¹H NMR.

ES-MS, m/z 296.2 (M+1)⁺.

1-D-(t-Butyl)glycinyl-4-(1-methylpiperidin-4-yl)piperidine.

Prepared from1-[Boc-D-(t-butyl)glycinyl]-4-(1-methyl-piperidin-4-yl)piperidine usingmethods substantially equivalent to those described in GeneralDeprotection Method 1.

¹H NMR.

ES-MS, m/z 296.2 (M+1)⁺.

1-D-Valinyl-4-(1-methylpiperidin-4-yl)piperazine Trihydrochloride.

1-(Boc-D-valinyl)-4-(1-methylpiperidin-4-yl)piperazine (8.9 g, 23 mmol)is dissolved in methanol (500 mL), and HCl gas is bubbled through thestirring solution for 10-15 min. After cooling to room temperature,diethyl ether (500 mL) is added, and the resulting solid is filtered anddried in vacuo to give 8.59 g (94%) of the title compound.

¹H NMR.

ES-MS, m/z 283.3 (M+1)⁺.

Analysis for C₁₅H₃₀N₃O₀·3.0 HCl0.3H₂O: Calcd: C, 45.43; H, 8.53; N,14.11; Found: C, 45.43; H, 8.25; N, 13.89.,

1-D-(t-Butyl)glycinyl-4-(1-methylpiperidin-4-yl)piperazine.

Prepared from1-[Boc-D-(t-butyl)glycinyl]-4-(1-methyl-piperidin-4-yl)piperazine usingmethods substantially equivalent to those described in GeneralDeprotection Method 1.

¹H NMR.

ES-MS, m/z 297.2 (M+1)⁺.

1-D-Isoleucinyl-4-(1-methylpiperidin-4-yl)piperazine.

Prepared from 1-(Boc-D-isoleucinyl)-4-(1-methylpiperidin-4-yl)piperazineusing methods substantially equivalent to those described in GeneralDeprotection Method 1. ES-MS, m/z 297.3 (M+1)⁺.

1-D-Leucinyl-4-(1-methylpiperidin-4-yl)piperazine.

Prepared from 1-(Boc-D-leucinyl)-4-(1-methylpiperidin-4-yl)piperazineusing methods substantially equivalent to those described in GeneralDeprotection Method 1.

¹H NMR.

ES-MS, m/z 297.3 (M+1)⁺.

EXAMPLE 1

1-(Indole-6-carbonyl-D-alaninyl)-4-(1-methylpiperidin-4-yl)-piperidinehydrochloride.

Prepared from 1-D-alaninyl-4-(1-methylpiperidin-4-yl)-piperidine andindole-6-carboxylic acid using methods substantially equivalent toGeneral Coupling Method 1. The HCl salt is prepared following GeneralSalt Formation Method 2.

¹H NMR.

ES-MS, m/z 397.1(M+1)⁺; 395.3 (M−1)⁻.

Analytical HPLC (Method 1): 98%, t_(r)=15.2 min.

EXAMPLE 2

1-(Indole-6-carbonyl-D-ethylglycinyl)-4-(1-methylpiperidin-4-yl)piperidinehydrochloride.

Prepared from 1-D-(ethyl)glycinyl-4-(1-methylpiperidin-4-yl)piperidinehydrochloride and indole-6-carboxylic acid using methods substantiallyequivalent to General Coupling Method 1. The HCl salt is preparedfollowing General Salt Formation Method 1.

¹H NMR.

ES-MS, m/z 411.2 (M+1)⁺; 409.2 (M−1)⁻.

Analysis for C₂₄H₃₄N₄O₂0.9 HCl2.0H₂O: Calcd: C, 60.13; H, 8.18; N,11.69; Cl, 6.66;. Found: C, 59.97; H, 7.97; N, 11.83; Cl, 6.27.

Analytical HPLC (Method 2): >95%, tr 17.4 min.

EXAMPLE 3

1-[Indole-6-carbonyl-D-(propyl)glycinyl]-4-(1-methylpiperidin-4-yl)piperidinehydrochloride.

Prepared from 1-D-propylglycinyl-4-(1-methylpiperidin-4-yl)piperidinehydrochloride and indole-6-carboxylic acid using methods substantiallyequivalent to General Coupling Method 1. The HCl salt is preparedfollowing Salt Formation Method 1.

¹H NMR.

ES-MS, m/z 425.2 (M+1)⁺; 423.2 (M−1)⁻.

Analysis for C₂₅H₃₆N₄O₂·0.9 HCl2.0H₂O: Calcd: C, 60.85; H, 8.35; N,11.36; Cl, 6.47; Found: C, 60.95; H, 7.98; N, 11.39; Cl, 5.93.

Analytical HPLC (Method 2): >98%, t_(r)=19.1 min.

EXAMPLE 4

1-[Indole-6-carbonyl-D-(butyl)glycinyl]-4-(1-methylpiperidin-4-yl)piperidinehydrochloride.

Prepared from 1-D-(butyl)glycinyl-4-(1-methylpiperidin-4-yl)piperidinehydrochloride and indole-6-carboxylic acid using methods substantiallyequivalent to General Coupling Method 1. The HCl salt is preparedfollowing General Salt Formation Method 1.

¹H NMR.

ES-MS, m/z 439.2 (M+1)⁺; 437.2 (M−1)⁻.

Analysis for C₂₆H₃₈N₄O₂·1 HCl2.2H₂O: Calcd: C, 60.67; H, 8.50; N, 10.89;Cl, 6.89; Found: C, 60.53; H, 8.13; N, 10.96; Cl, 6.67.

Analytical HPLC (Method 2): >95%, t_(r)=21.1 min.

EXAMPLE 5

1-(4-Methoxybenzoyl-D-valinyl)-4-(1-methylpiperidin-4-yl)-piperidine.

1-D-Valinyl-4-(1-methylpiperidin-4-yl)piperidine dihydrochloride (15.6g, 44.1 mmol) is slurried in anhydrous CH₂Cl₂ (400 mL); and to theslurry is added triethylamine (19.7 mL, 141.0 mmol), followed byp-anisoyl chloride (7.9 mL, 52.9 mmol). The reaction is allowed to stirfor about 30 min and then quenched with the addition of saturated NaHCO₃(300 mL), followed by the separation of the phases. The organic layer iswashed with saturated NaHCO₃ (2×300 mL), dried over Na₂SO₄, filtered andconcentrated in vacuo. The residue is purified by chromatography oversilica gel, eluting with 2.5% (2 M NH₃ in MeOH)/CHCl₃ to give 16.3 g(89%) of the title compound as a white foam.

¹H NMR (DMSO-d₆) δ 8.25 (m, 1H), 7.88 (m, 2H), 6.97 (m, 2H), 4.71 (m,1H), 4.45 (m, 1H), 4.21 (m, 1H), 3.80 (s, 3H), 2.96 (m, 1H), 2.74 (m,2H), 2.13 (m, 1H), 2.10 (m, 3H), 1.8-1.5 (bm, 6H), 1.3-0.9 (bm, 7H),0.89 (m, 6H).

ES-MS, m/z 416.4 (M+1)⁺.

[α]D²⁰=−41.12 (c=0.01 MeOH).

EXAMPLE 6

1-(4-Methoxybenzoyl-D-valinyl)-4-(1-methylpiperidin-4-yl)-piperidineHydrochloride.

To a solution of1-(4-methoxybenzoyl-D-valinyl)-4-(1-methylpiperidin-4-yl)piperidine(14.75 g, 35.5 mmol) in anhydrous CH₂Cl₂ (400 mL) cooled to 0° C. isadded HCl in Et₂O (1 M, 35.9 mL, 35.9 mmol). After approximately 5 min,the solvent is removed in vacuo to give 16.9 g (quantitative) of thetitle compound as a white foam.

¹H NMR (DMSO-d₆) δ 10.0 (bs, 1H), 8.27 (m, 1H), 7.88 (m, 2H), 6.98 (m,2H), 4.71 (m, 1H), 4.46 (m, 1H), 4.23 (m, 1H), 3.81 (s, 3H), 3.43 (s,3H), 3.37 (m, 2H), 3.00 (m, 1H), 2.83 (m, 2H), 2.68 (m, 2H), 2.15 (bm,1H), 1.9-1.6 (m, 4H), 1.5-1.2 (bm, 4H), 1.07 (m, 1H), 0.91 (m, 3H), 0.87(m, 3H).

ES-MS, m/z 416.4 (M+1)⁺.

[α]_(D) ²⁰=−42.72 (c=0.01 MeOH).

Analysis for C₂₄H₃₇N₃O₃1.0 HCl-1.2H₂O·0.25 CH₂Cl₂: Calcd: C 59.93; H8.48; N 8.65; Cl 9.12. Found: C 59.67; H 8.38; N 8.52; Cl 9.35.

EXAMPLE 7

1-(4-Methoxybenzoyl-D-valinyl)-4-(1-methylpiperidin-4-yl)-piperidineMethanesulfonic Acid Salt.

The mesylate salt is prepared by dissolving the free base in EtOAc(example concentration, 416 mg/10 mL). Separately, methanesulfonic acidis dissolved in EtOAc (example concentration, 96 mg/2 mL). Themethanesulfonic acid solution is added to the base solution and theinitial mixture becomes hazy. The vial is seeded, and the seeds stick tosides as the material starts to oil. The oil is allowed to sit overnightand is crystalline in the morning. The crystals are washed with a smallamount of EtOAc and air dried. The yield is 460 mg (about 90%) based ona monomesylate salt, as later confirmed. The material so obtained iscrystalline by photomicroscopy revealing birefringence. Analysis by DSC(differential scanning calorimetry) and TGA (thermal gravimetricanalysis) indicated that the monomesylate salt had a melting point withits endotherm peak at 185° C., with 0.9% volatiles lost at 25-150° C.and 1.9% volatiles released at 150-185° C.

On larger scale, the following procedure is convenient: To a wellstirred solution of the free base (125 g) in ethyl acetate (2.5 L) isadded a solution of methanesulfonic acid in ethyl acetate (0.5 L)dropwise. After addition, the mixture is stirred 4 h at room temperaturebefore the crystalline product is filtered, washed with ethyl acetate(0.5 L), and dried under vacuum (35° C., 24 h) to provide the title saltas a white crystalline solid (139.5 g, 90.0%).

melting point 182-183° C.

[α]_(D) ²⁰=−43.0 (c=0.416H₂O).

enatiomeric excess greater than 99% by chiral HPLC

EXAMPLE 8

1-(Indole-6-carbonyl-D-valinyl)-4-(1-methylpiperidin-4-yl)-piperidine.

6-Carboxyindole (4.0 g, 24.8 mmol) and1-(D-valinyl)-4-(1-methylpiperidin-4-yl)piperidine (10.1 g, 27.8 mmol)are slurried in anhydrous CH₂Cl₂ (250 mL) and cooled to −15° C. To thisslurry is added diethyl cyanophosphonate (4.5 mL, 29.8 mmol), followedby TEA (11.1 mL, 79.4 mmol) maintaining the temperature below −10° C.The reaction is allowed to warm to room temperature overnight. Thefollowing morning, if TLC indicates starting material still present,additional 6-carboxyindole (0.5 g, 3.1 mmol) and diethylcyanophosphonate (0.5 mL, 3.3 mmol) are added, followed by TEA (1.0 mL,7.2 mmol). After stirring for an additional 2 h, the reaction isquenched with the addition of brine (100 mL), forming an insoluble brownoil on the sides of the flask. The layers are separated and the aqueouslayer is extracted with CH₂Cl₂ (3×100 mL). The organic layers arecombined, dried over Na₂SO₄, filtered and concentrated. The crudematerial is then purified using chromatography over silica gel, with 5%(2 M NH₃ in MeOH)/CHCl₃ as the eluent, to give 9.7 g (92%) of the titlecompound as a light yellow foam.

¹H NMR (DMSO-d₆) δ 11.35 (s, 1H), 8.22 (m, 1H), 7.97 (s, 1H), 7.55 (s,2H), 7.49 (s, 1H), 6.48 (s, 1H), 4.76 (m, 1H), 4.46 (m, 1H), 4.23 (m,1H), 2.99 (m, 1H), 2.73 (m, 2H), 2.16 (m, 1H), 2.09 (m, 3H), 1.8-1.5 (m,6H), 1.4-1.0 (m, 6H), 0.91 (m, 6H).

ES-MS, m/z 425.1 (M+1)⁺.

Examination by photomicroscopy showed the free base to be obtained as abirefringent crystalline solid which was shown by analysis by DSC(differential scanning calorimetry) and TGA (thermal gravimetricanalysis) to have a melting point with its endotherm peak at 196° C.,and only about 0.4% volatiles.

EXAMPLE 9

1-(Indole-6-carbonyl-D-valinyl)-4-(1-methylpiperidin-4-yl)piperidineHydrochloride

1-(Indole-6-carbonyl-D-valinyl)-4-(1-methylpiperidin-4-yl)piperidine(3.4 g, 8.03 mmol) is suspended in 0.2 N aqueous HCl (40.2 mL, 8.03mmol) and sonicated. The resulting solution is lyophilized to give 3.64g (98%) of the title compound.

¹H NMR

ES-MS, m/z 425.3 (M+1)⁺.

Analysis for C₂₅H₃₆N₄O₃1.1 HCl1.0H₂O: Calcd: C, 62.20; H, 8.16; N,11.61; Cl, 8.08; Found: C, 61.91; H, 7.80; N, 11.51; Cl, 7.79.

HPLC Analysis (Method 1): 99% t_(r)=20.80 min.

EXAMPLE 10

1-(3-Methylindole-6-carbonyl-D-valinyl)-4-(1-methylpiperidin-4-yl)piperidineHydrochloride.

Prepared from 1-D-valinyl-4-(1-methylpiperidin-4-yl)-piperidinehydrochloride and 3-methylindole-6-carboxylic acid using methodssubstantially equivalent to General Coupling Method 2. The HCl salt isprepared following General Salt Formation Method 2.

¹H NMR.

ES-MS, m/z 439.0 (M+1)⁺.

Analysis for C₂₆H₃₈N₄O₂1.2 HCl-0.9H₂O: Calcd: C, 62.63; H, 8.29; N,11.24; Cl, 8.53; Found: C, 62.46; H, 8.34; N, 11.03; Cl, 8.96.

Analytical HPLC (Method 1): 100%, t_(r)=25.3 min.

EXAMPLE 11

1-(3-Chloroindole-6-carbonyl-D-valinyl)-4-(1-methylpiperidin-4-yl)piperidineHydrochloride.

Prepared from 1-D-valinyl-4-(1-methylpiperidin-4-yl)-piperidinehydrochloride and 3-chloroindole-6-carboxylic acid using methodssubstantially equivalent to General Coupling Method 1. The HCl salt isprepared following General Salt Formation Method 2.

¹H NMR.

ES-MS, m/z 459.0 (M+1)⁺.

Analysis for C₂₅H₃₅N₄O₂·1.1 HCl-0.7H₂O: Calcd: C, 58.68; H, 7.39; N,10.95; Cl, 14.55;. Found: C, 58.55; H, 7.41; N, 10.43; Cl, 14.46.

Analytical HPLC (Method 1): 100%, tr 27.8 min.

EXAMPLE 12

1-(5-Chloroindole-2-carbonyl-D-valinyl)-4-(1-methylpiperidin-4-yl)piperidineHydrochloride.

Prepared from 1-D-valinyl-4-(1-methylpiperidin-4-yl)-piperidinehydrochloride and 5-chloroindole-2-carboxylic acid using methodssubstantially equivalent to General Coupling Method 2. Purified andconverted to the hydrochloride salt from by preparative reverse phaseHPLC (YMC ODSA C18 5μ column, 5-95% CH₃CN in H₂O with 0.01% HCl) to givethe HCl salt.

¹H NMR.

ES-MS, m/z 459.1 (M+1)⁺.

Analysis for C₂₅ClH₃₅N₄O₂·0.8 HCl2.2H₂O: Calcd: C, 56.89; H, 7.68; N,10.62; Cl, 12.09; Found: C, 56.98; H, 6.94; N, 10.45; Cl, 12.13.

-   -   Analytical HPLC (Method 1): 100%, t_(r)=25.3 min.

EXAMPLE 13

1-(Indole-6-carbonyl-D-leucinyl)-4-(1-methylpiperidin-4-yl)piperidineHydrochloride.

Prepared from 1-D-leucinyl-4-(1-methylpiperidin-4-yl)-piperidine andindole-6-carboxylic acid using methods substantially equivalent to thosedescribed in General Coupling Method 1. The salt is prepared usingGeneral Salt Formation Method 2.

¹H NMR.

FD-MS, m/z 438.3 M+.

Analysis for C₂₆H₃₈N₄O₂·1.1 HCl·1.4H₂O: Calcd: C, 61.97; H, 8.38; N,11.12; Cl, 7.74; Found: C, 62.00; H, 8.00; N, 11.18; Cl, 7.58.

Analytical HPLC (Method 1): >95%, t_(r)=24.43 min.

EXAMPLE 14

1-[Indole-6-carbonyl-D-(t-butyl)glycinyl]-4-(1-methylpiperidin-4-yl)piperidineHydrochloride.

Prepared from1-[D-(t-butyl)glycinyl]-4-(1-methyl-piperidin-4-yl)piperidine andindole-6-carboxylic acid using methods substantially equivalent to thosedescribed in General Coupling Method 1. The HCl salt is preparedfollowing General Salt Formation Method 2.

¹H NMR.

ES-MS, m/z 439.1 (M+1)⁺.

Analysis for C₂₆H₃₈N₄O₂·1.1 HCl1.7H₂O: Calcd: C, 61.31; H, 8.41; N,11.00; Found: C, 61.43; H, 8.28; N, 10.65.

Analytical HPLC (Method 1): 100%, t_(r)=23.86 min.

EXAMPLE 15

1-(Indole-6-carbonyl-D-valinyl)-4-(1-methylpiperidin-4-yl)-piperazine.

Prepared from 1-D-valinyl-4-(1-methylpiperidin-4-yl)-piperazine andindole-6-carboxylic acid using methods substantially equivalent to thosedescribed in General Coupling Method 1. The HCl salt is preparedfollowing General Salt Formation Method 2.

¹H NMR.

ES-MS, m/z 426.2 (M+1)⁺.

Analysis for C₂₄H₃₅N₅O₂·1.4 HCl2.1H₂O: Calcd: C, 56.03; H, 7.95; N,13.61; Found:, C, 56.05; H, 7.57; N, 13.24.

Analytical HPLC (Method 1): 100%, t_(r)=16.55 min.

EXAMPLE 16

1-(3-Chloroindole-6-carbonyl-D-valinyl)-4-(1-methylpiperidin-4-yl)piperazineHydrochloride.

Prepared from 1-D-valinyl-4-(1-methylpiperidin-4-yl)-piperazinehydrochloride and 3-chloroindole-6-carboxylic acid using methodssubstantially equivalent to General Coupling Method 1. The HCl salt isprepared following General Salt Formation Method 1.

¹H NMR.

ES-MS, m/z 460.2 (M+1)⁺.

Analysis for C₂₄H₃₄N₅O₂C₁₋₂ HCl-1.1H₂O: Calcd: C, 56.05; H, 7.20; N,13.38; Cl, 14.90; Found: C, 56.05; H, 7.08; N, 13.35; Cl, 14.82.

Analytical HPLC (Method 3): 97%, t_(r)=19.3 min.

EXAMPLE 17

1-(3-Methylindole-6-carbonyl-D-valinyl)-4-(1-methylpiperidin-4-yl)piperazineHydrochloride.

Prepared from 1-D-valinyl-4-(1-methylpiperidin-4-yl)-piperazine and3-methylindole-6-carboxylic acid using methods substantially equivalentto General Coupling Method 1. The product was purified and converted tothe hydrochloride salt by preparative reverse phase HPLC (C18, 90:10 to50:50-0.1 N HCl:CH₃CN).

¹H NMR.

ES-MS, m/z 440.3 (M+1)⁺.

Analytical HPLC (Method 1): 100%, t_(r)=21.5 min.

EXAMPLE 18

1-(5-Chloroindole-2-carbonyl-D-valinyl)-4-(1-methylpiperidin-4-yl)piperazineHydrochloride.

To a solution of D-valinyl-4-(1-methylpiperidin-4-yl)-piperazinehydrochloride (3.5 g, 8.93 mmol) and triethylamine (4.4 mL, 31.3 mmol)in dichloromethane (90 mL) is added HOBt (1.33 g, 9.82 mmol) followed byEDCI (1.9 g, 9.82 mmol). After stirring overnight the mixture isconcentrated in vacuo. The residue is then chromatographed over silicagel, eluting with a gradient of 0-15% (2 N ammonia/methanol) inchloroform. The product containing fractions are combined andconcentrated in vacuo to give 3.97 g (97%) of the free base of the titlecompound.

The free base (2.183 g, 4.75 mmol) is dissolved in 0.2 N HCl (23.8 mL)and lyophilized to give the title compound.

¹H NMR.

ES-MS, m/z 460.2 (M+1)⁺.

Analysis for C₂₄H₃₄N₅O₂C₁₋₂ HCl 1.6H₂O: Calcd: C, 54.12; H, 7.27; N,13.15; Cl, 14.65; Found: C, 54.02; H, 7.13; N, 13.04; Cl, 14.65.

Analytical HPLC (Method 3): 97.7%, t_(r)=22.9 min.

EXAMPLE 19

1-(Indole-2-carbonyl-D-valinyl)-4-(1-methylpiperidin-4-yl)-piperazineHydrochloride.

Prepared from l-D-valinyl-4-(1-methylpiperidin-4-yl)-piperazinehydrochloride and indole-2-carboxylic acid using methods substantiallyequivalent to General Coupling Method 1. The HCl salt is preparedfollowing General Salt Formation Method 1.

¹H NMR.

ES-MS, m/z 426.3 (M+1)⁺.

Analysis for C₂₄H₃₅N₅O₂-2 HCl-1.6H₂O: Calcd: C, 57.45; H, 8.06; N,13.96; Cl, 7.77; Found: C, 57.37; H, 7.39; N, 13.68; Cl, 8.15.

EXAMPLE 20

1-[Indole-6-carbonyl-D-(t-butyl)glycinyl]-4-(1-methylpiperidin-4-yl)piperazineHydrochloride.

Prepared from l-D-(t-butyl)glycinyl-4-(1-methylpiperidin-4-yl)piperazineand indole-6-carboxylic acid using methods substantially equivalent tothose described in General Coupling Method 1. The HCl salt is preparedfollowing General Salt Formation Method 2.

¹H NMR.

ES-MS, m/z 440.3 (M+1)⁺.

Analysis for C₂₅H₃₇NsO-2.1 HCl 1.9H₂O: Calcd: C, 54.55; H, 7.86; N,12.72; Cl, 13.53; Found: C, 54.39; H, 7.72; N, 12.34; Cl, 13.85.

Analytical HPLC (Method 1): 94%, t_(r)=17.07 min.

EXAMPLE 21

1-(Indole-6-carbonyl-D-isoleucinyl)-4-(1-methylpiperidin-4-yl)-piperazineHydrochloride.

Prepared from 1-D-isoleucinyl-4-(1-methylpiperidin-4-yl)-piperazine andindole-6-carboxylic acid using methods substantially equivalent to thosedescribed in General Coupling Method 1. The HCl salt is preparedfollowing General Salt Formation Method 2.

¹H NMR.

ES-MS, m/z 440.3 (M+1)⁺.

Analysis for C₂₅H₃₇N₅O-2.3 HCl 1.8H₂O: Calcd: C, 54.19; H, 7.77; N,12.64; Cl, 14.72; Found: C, 53.91; H, 7.37; N, 12.02; Cl, 14.90.Analytical HPLC (Method 1): 95%, t_(r)=18.08 min.

EXAMPLE 22

1-(Indole-6-carbonyl-D-leucinyl)-4-(1-methylpiperidin-4-yl)-piperazineHydrochloride.

Prepared from 1-D-leucinyl-4-(1-methylpiperidin-4-yl)-piperazine andindole-6-carboxylic acid using methods substantially equivalent to thosedescribed in General Coupling Method 1. The HCl salt is preparedfollowing General Salt Formation Method 2.

¹H NMR.

ES-MS, m/z 440.2 (M+1)⁺.

Analysis for C₂₅H₃₇N₅O·1.1 HCl3.0H₂O: Calcd: C, 56.25; H, 8.33; N,13.12; Cl, 7.31; Found: C, 56.34; H, 7.64; N, 12.75; Cl, 7.06.

Analytical HPLC (Method 1): 100%, t_(r)=18.72 min

Further information for following examples:

HPLC Analysis (Method A): Waters Symmetry, C18 (4.6×250 mm) column. Theelution system consisted of linear gradient from 95:5 (0.1% TFA inH₂O)/(0.1% TFA in CH₃CN) to 5:95 (0.1% TFA in H₂O)/(0.1% TFA in CH₃CN)over 20 min, followed by 5:95 (0.1% TFA in H₂O)/(0.1% TFA in CH₃CN)isocratic over 10 min. The flow rate was 1 mL/min. UV Detection wasperformed at 254 nm unless otherwise noted.

API-MS (atmospheric pressure chemical ionization mass spectra) wereobtained on a PESciex API 150EX with a heated nebulizer and nitrogen asthe reagent gas in positive ion mode.

CI-MS (Chemical ionization mass spectra) were obtained on a Shimadzu5000 direct insertion mass spectrometer in chemical ionization modeutilizing methane as the reagent gas.

The following further abbreviations are used: CMA(chloroform:methanol:concentrated ammonium hydroxide 80:18:2), DEPC(diethyl cyanophosphonate).

General Coupling Method 4: The amine or amine hydrochloride salt (1 eq)and triethylamine (1-3 eq) are dissolved in dichloromethane (0.1-0.5 M)and an acid chloride (about 1.1 eq) is added. After stirring overnight,the reaction mixture is quenched with saturated NaHCO₃ solution. Theaqueous layer is extracted twice with dichloromethane, and the combinedorganic layers are dried over Na₂SO₄, filtered and concentrated invacuo. If necessary, the product is purified by chromatography oversilica gel, eluting with a gradient of 0-10% 2 N ammonia/methanol indichloromethane. The product-containing fractions are then combined andconcentrated in vacuo. Alternatively, the crude product is purified byreverse phase HPLC on C-18 using a 5 to 95% gradient of acetonitrile inH₂O with 0.01% HCl over 12 min. Freeze-drying gives the product as itsHCl salt.

EXAMPLE 23

1-(3-Chloroindole-6-carbonyl-D-ethylglycinyl)-4-(1-methyl-piperidin-4-yl)piperidineHydrochloride.

Prepared from 1-D-ethylglycinyl-4-(1-methylpiperidin-4-yl)piperidinedihydrochloride and 3-chloroindole-6-carboxylic acid using methodssubstantially equivalent to General Coupling Method 1. The HCl salt isprepared following General Salt Formation Method 1.

¹H NMR.

ES-MS, m/z 445.2 (M+1)⁺.

Analysis for C₂₄H₃₃N₄O₂Cl·1.0 HCl 1.6H₂O: Calcd: C 56.49; H 7.35; N10.98; Cl 13.90; Found: C 56.58; H 6.95; N 10.93; Cl 13.44.

Analytical HPLC (Method 2): >97%, t_(r)=20.7 min.

EXAMPLE 24

1-(5-Chloroindole-2-carbonyl-D-ethylglycinyl)-4-(1-methyl-piperidin-4-yl)piperidineHydrochloride.

Prepared from 1-D-ethylglycinyl-4-(1-methylpiperidin-4-yl)piperidinedihydrochloride and 5-chloroindole-2-carboxylic acid using methodssubstantially equivalent to General Coupling Method 1. The HCl salt isprepared following General Salt Formation Method 1.

¹H NMR.

ES-MS, m/z 445.2 (M+1)⁺.

Analysis Calcd. For C₂₄H₃₃N₄O₂Cl·1.0 HCl·2.1H₂O: Calcd: C 55.51; H 7.41;N 10.79; Cl 13.66; Found: C 55.61; H 6.90; N 10.77; Cl 13.57.

Analytical HPLC (Method 2): >90%, t_(r)=22.3 min.

EXAMPLE 25

1-(4-Methoxybenzoyl-D-ethylglycinyl)-4-(1-methylpiperidin-4-yl)piperidineHydrochloride.

Prepared from 1-D-ethylglycinyl-4-(1-methylpiperidin-4-yl)piperidinedihydrochloride and 4-methoxybenzoyl chloride using methodssubstantially equivalent to General Coupling Method 4. Purification bythe reverse phase chromatography option gives the HCl salt.

¹H NMR.

ES-MS, m/z 402.3 (M+1)⁺.

Analysis for C₂₃H₃₅N₃O₃·1.0 HCl-1.4H₂O: Calcd: C 59.63; H 8.44; N 9.07;Found: C 59.75; H 8.24; N 8.91.

EXAMPLE 26

1-[(3-Fluoro-4-methoxybenzoyl)-D-ethylglycinyl)]-4-(1-methylpiperidin-4-yl)piperidineHydrochloride.

Prepared from l-D-ethylglycinyl-4-(1-methylpiperidin-4-yl)piperidinedihydrochloride and 3-fluoro-4-methoxybenzoic acid using methodssubstantially equivalent to General Coupling Method 1 with reverse phasepurification as described in General Coupling Method 4 to give the HClsalt.

¹H NMR.

ES-MS, m/z 420.3 (M+1)⁺.

Analysis for C₂₃H₃₄N₃O₃F·1.7 HCl·1.0H₂O: Calcd: C 55.30; H 7.61; N 8.41;Found: C 55.22; H 7.22; N 8.42.

EXAMPLE 27

1-(6-Chlorobenzo[b]thiophene-2-carbonyl-D-ethylglycinyl)-4-(1-methylpiperidin-4-yl)piperidineHydrochloride.

Prepared from 1-D-ethylglycinyl-4-(1-methylpiperidin-4-yl)piperidinedihydrochloride and 6-chlorobenzo[b]thiophene-2-carboxylic acid usingmethods substantially equivalent to General Coupling Method 1 withreverse phase purification as described in General Coupling Method 4 togive the HCl salt.

¹H NMR.

ES-MS, m/z 462.2 (M+1)⁺.

Analysis for C₂₄H₃₂N₃O₂SCl·1.0 HCl·1.2H₂O: Calcd: C 55.42; H 6.86; N8.08; Found: C 55.50; H 6.65; N 8.04.

EXAMPLE 28

1-[3-Chloroindole-6-carbonyl-D-propylglycinyl]-4-(1-methylpiperidin-4-yl)piperidineHydrochloride.

Prepared from 1-D-propylglycinyl-4-(1-methylpiperidin-4-yl)piperidinedihydrochloride and 3-chloroindole-6-carboxylic acid using methodssubstantially equivalent to General Coupling Method 1. The HCl salt isprepared following General Salt Formation Method 1.

¹H NMR.

ES-MS, m/z 459.3 (M+1)⁺.

Analysis for C₂₅H₃₅N₄O₂Cl·1.0 HCl·2.3H₂O: Calcd: C 55.92; H 7.62; N10.44; Cl 13.21; Found: C 55.84; H 6.95; N 10.35; Cl 13.16.

Analytical HPLC (Method 2): >90%, t_(r)=22.3 min.

EXAMPLE 29

1-[5-Chloroindole-2-carbonyl-D-propylglycinyl]-4-(1-methylpiperidin-4-yl)piperidineHydrochloride.

Prepared from 1-D-propylglycinyl-4-(1-methylpiperidin-4-yl)piperidinedihydrochloride and 5-chloroindole-2-carboxylic acid using methodssubstantially equivalent to General Coupling Method 1. The HCl salt isprepared following General Salt Formation Method 1.

¹H NMR.

ES-MS, m/z 459.3 (M+1)⁺.

Analysis for C₂₅H₃₅N₄O₂Cl·0.9 HCl·1.9H₂O: Calcd: C 57.08; H 7.61; N10.65; Cl 12.81; Found: C 57.09; H 6.98; N 10.71; Cl 12.74.

Analytical HPLC (Method 2): >91%, t_(r)=24.0 min.

EXAMPLE 30

1-[4-Methoxybenzoyl-D-propylglycinyl]-4-(1-methylpiperidin-4-yl)piperidineHydrochloride.

Prepared from 1-D-propylglycinyl-4-(1-methylpiperidin-4-yl)piperidinedihydrochloride and 4-methoxybenzoyl chloride using methodssubstantially equivalent to General Coupling Method 4. Purification bythe reverse phase chromatography option gives the HCl salt.

¹H NMR.

ES-MS, m/z 416.3 (M+1)⁺.

Analysis for C₂₄H₃₇N₃O₃·1.0 HCl·1.4H₂O: Calcd: C 60.40; H 8.62; N 8.81;Found: C 60.49; H 8.38; N 8.78.

EXAMPLE 31

1-[(3-Fluoro-4-methoxybenzoyl)-D-propylglycinyl]-4-(1-methylpiperidin-4-yl)piperidineHydrochloride.

Prepared from 1-D-propylglycinyl-4-(1-methylpiperidin-4-yl)piperidinedihydrochloride and 3-fluoro-4-methoxybenzoic acid using methodssubstantially equivalent to General Coupling Method 1 with reverse phasepurification as described in General Coupling Method 4 to give the HClsalt.

¹H NMR.

ES-MS, m/z 434.3 (M+1)⁺.

Analysis for C₂₄H₃₆N₃O₃F·1.1 HCl·1.6H₂O: Calcd: C 57.36; H 8.08; N 8.36;Found: C 57.36; H 7.80; N 8.33.

EXAMPLE 32

1-[6-Chlorobenzo[b]thiophene-2-carbonyl-D-propylglycinyl]-4-(1-methylpiperidin-4-yl)piperidineHydrochloride.

Prepared from 1-D-propylglycinyl-4-(1-methylpiperidin-4-yl)piperidinedihydrochloride and 6-chlorobenzo[b]thiophene-2-carboxylic acid usingmethods substantially equivalent to General Coupling Method 1. The HClsalt is prepared following General Salt Formation Method 1 with reversephase purification as described in General Coupling Method 4 to give theHCl salt.

¹H NMR.

ES-MS, m/z 476.2 (M+1)⁺.

Analysis for C₂₅H₃₄N₃O₂SCl·1.0 HCl·1.3H₂O: Calcd: C 56.02; H 7.07; N7.84; Found: C 55.95; H 6.79; N 7.74.

EXAMPLE 33

1-[3-Chloroindole-6-carbonyl-D-butylglycinyl]-4-(1-methylpiperidin-4-yl)piperidineHydrochloride.

Prepared from 1-D-butylglycinyl-4-(1-methylpiperidin-4-yl)piperidinedihydrochloride and 3-chloroindole-6-carboxylic acid using methodssubstantially equivalent to General Coupling Method 1. The HCl salt isprepared following General Salt Formation Method 1.

¹H NMR.

ES-MS, m/z 473.3 (M+1)⁺.

Analysis for C₂₆H₃₇N₄O₂Cl·0.9 HCl·2.0H₂O: Calcd: C 57.63; H 7.79; N10.34; Cl 12.43; Found: C 57.48; H 7.49; N 10.41; Cl 12.36. AnalyticalHPLC (Method 2): >96%, t_(r)=23.9 min.

EXAMPLE 34

1-[5-Chloroindole-2-carbonyl-D-butylglycinyl]-4-(1-methylpiperidin-4-yl)piperidineHydrochloride.

Prepared from 1-D-butylglycinyl-4-(1-methylpiperidin-4-yl)piperidinedihydrochloride and 5-chloroindole-2-carboxylic acid using methodssubstantially equivalent to General Coupling Method 1. The HCl salt isprepared following General Salt Formation Method 1.

¹H NMR.

ES-MS, m/z 473.3 (M+1)⁺.

Analysis Calcd. For C₂₆H₃₇N₄O₂Cl·0.9 HCl·1.9H₂O: Calcd: C 57.82; H 7.78;N 10.37; Cl 12.47; Found: C 57.93; H 7.24; N 10.54; Cl 12.20. AnalyticalHPLC (Method 2): >95%, t_(r)=25.5 min.

Preparation of Intermediates

1-N-Boc-D-ethylglycinyl-4-(1-methylpiperidin-4-yl)piperazine.

Prepared from Boc-D-ethylglycine and4-(1-methylpiperidin-4-yl)piperazine using methods substantiallyequivalent to General Coupling Method 1.

¹H NMR.

ES-MS, m/z 369.3 (M+1)⁺.

Analysis for C₁₉H₃₆N₄O₃·0.3H₂O: Calcd: C 61.03; H 9.87; N 14.98; Found:C 61.02; H 9.39; N 14.87.

1-N-Boc-D-propylglycinyl-4-(1-methylpiperidin-4-yl)piperazine.

Prepared from Boc-D-propylglycine and4-(1-methyl-piperidin-4-yl)piperazine using methods substantiallyequivalent to General Coupling Method 1.

¹H NMR.

ES-MS, m/z 383.3 (M+1)⁺.

1-N-Boc-D-butylglycinyl-4-(1-methylpiperidin-4-yl)piperazine.

Prepared from Boc-D-butylglycine and4-(1-methylpiperidin-4-yl)piperazine using methods substantiallyequivalent to General Coupling Method 1.

¹H NMR.

ES-MS, m/z 397.3 (M+1)⁺.

Analysis for C₂₁H₄₀N₄O₃·0.3H₂O: Calcd: C 62.75; H 10.18; N 13.94; Found:C 62.89; H 9.71; N 13.88.

N-Boc-D-Propargylglycine.

Method A-1: A solution of di-tert-butyl dicarbonate (5.7 g, 26.4 mmol)in 1,4-dioxane (20 mL) is added into an efficiently stirred solution ofD-propargylglycine (2.5 g, 22.1 mmol) and NaOH (1.86 g, 46.5 mmol) inwater (45 mL) at 0° C. The mixture is stirred 30 min at 0° C. and 3.5 hat room temperature. The mixture is concentrated to one half volume at35° C. under vacuum. The solution is cooled in an ice/water bath andacidified to pH 2-3 with potassium hydrogen sulfate solution (2 N). Themixture is extracted with ethyl acetate; and the organic layer is washedwith water, dried over anhydrous sodium sulfate, and concentrated undervacuum to provide the titled compound (4.6 g, 97%).

¹H NMR (CDCl₃).

APCI-MS, m/e=214 (M+1).

1-(N-Boc-D-Propargylglycinyl)-4-(1-methylpiperidin-4-yl)-piperazine.

Method B-1: To a mixture of N-Boc-D-propargylglycine (4.6 g, 21.6 mmol),4-(1-methylpiperidin-4-yl)piperazine (3.91 g, 21.6 mmol), HOBt (2.88 g,21.6 mmol), EDCI (5.3 g, 28 mmol), CH₂Cl₂ (250 mL) is addeddiisopropylethylamine (7.9 mL, 43.2 mmol) at room temperature. Themixture is stirred overnight. The mixture is diluted withdichloromethane and water. The layers are separated; and the organiclayer is dried over anhydrous sodium sulfate, filtered, and concentratedunder vacuum. The residue is purified by chromatography on silica gelwith hexanes/CMA to provide the titled compound (3.4 g, 41%).

¹H NMR (CDCl₃).

APCI-MS, m/e=379 (M+1).

N-Boc-D-Allylglycine.

Using methods substantially equivalent to that described in Method A-1,the titled compound is prepared from D-allylglycine and di-tert-butyldicarbonate (81%).

¹H NMR (CDCl₃).

APCI-MS, m/e=216 (M+1).

1-(N-Boc-D-Allylglycinyl)-4-(1-methylpiperidin-4-yl)piperazine.

Using methods substantially equivalent to that described in Method B-1,the titled compound is prepared from N-Boc-D-allylglycine and4-(1-methylpiperidin-4-yl)piperazine (31%).

¹H NMR (CDCl₃).

APCI-MS, m/e=380 (M+1).

1-D-Ethylglycinyl-4-(1-methylpiperidin-4-yl)piperazine Trihydrochloride.

Prepared from 1-Boc-D-ethylglycinyl-4-(1-methylpiperidin-4-yl)piperazineusing methods substantially equivalent to those described in GeneralDeprotection Method 2.

¹H NMR.

ES-MS, m/z 269.2 (M+1)⁺.

Analysis for C₁₄H₂₈N₄O·3HCl: Calcd: C 44.51; H 8.27; N 14.83; Cl 28.15;Found: C 44.48; H 8.02; N 14.79; Cl 27.77.

1-D-Propylglycinyl-4-(1-methylpiperidin-4-yl)piperazineTrihydrochloride.

Prepared from1-Boc-D-propylglycinyl-4-(1-methylpiperidin-4-yl)piperazine usingmethods substantially equivalent to those described in GeneralDeprotection Method 2.

¹H NMR.

ES-MS, m/z 283.3 (M+1)⁺.

Analysis for C₁₅H₃₀N₄O·3 HCl·0.2H₂O: Calcd: C 45.68; H 8.28; N 14.21; Cl26.97; Found: C 45.86; H 8.29; N 14.11; Cl 26.85.

1-D-Butylglycinyl-4-(1-methylpiperidin-4-yl)piperazine Trihydrochloride.

Prepared from 1-Boc-D-butylglycinyl-4-(1-methylpiperidin-4-yl)piperazineusing methods substantially equivalent to those described in GeneralDeprotection Method 2.

¹H NMR.

ES-MS, m/z 297.3 (M+1)⁺.

Analysis for C₁₆H₃₂N₄O·2.9 HCl·1.2H₂O: Calcd: C 45.45; H 8.65; N 13.25;Cl 24.32; Found: C 45.23; H 8.27; N 13.00; Cl 24.39.

1-(D-Propargylglycinyl)-4-(1-methylpiperidin-4-yl)piperazine.

Method C-1: A mixture of1-(1-Boc-D-propargylglycinyl)-4-(1-methylpiperidin-4-yl)piperazine (3.4g, 9 mmol), methanol (50 mL), and anisole (15 mL) is cooled to 0° C.Concentrated HCl (20 mL) is added dropwise. The mixture is stirred 2 hat room temperature. The mixture is concentrated under vacuum. Theresidue is dissolved in methanol and applied to SCX resin (activatedwith 5% acetic acid in methanol and washed with methanol), washed withmethanol, and eluted with saturated ammonia in methanol. The productfraction is concentrated under vacuum to provide the titled compound asan oil (2.1 g, 84%).

¹H NMR (CDCl₃).

APCI-MS, m/e=279 (M+1).

1-(D-Allylglycinyl)-4-(1-methylpiperidin-4-yl)piperazine.

Method C-2: To a stirred solution of1-(Boc-D-allylglycinyl)-4-(1-methylpiperidin-4-yl)piperazine (2.2 g) indichloromethane (40 mL) at 0° C. is added trifluoroacetic acid (20 mL).The solution is allowed to warm to room temperature over 3 h. Thesolvents are removed under vacuum. The residue is dissolved in methanoland applied to a SCX column (pre-washed with 5% acetic acid in methanoland methanol), washed with methanol and eluted with saturated ammonia inmethanol. The product fraction is collected and concentrated undervacuum to provide the titled compound (1.6 g, quantitative) as a yellowoil.

¹H NMR (CD₃OD).

APCI-MS, m/e 281 (C₁₅H₂₈N₄O+1).

EXAMPLE 35

1-(Indole-6-carbonyl-D-ethylglycinyl)-4-(1-methylpiperidin-4-yl)piperazineHydrochloride.

Prepared from 1-D-ethylglycinyl-4-(1-methylpiperidin-4-yl)piperazinetrihydrochloride and indole-6-carboxylic acid using methodssubstantially equivalent to General Coupling Method 1. The HCl salt isprepared following General Salt Formation Method 1.

¹H NMR.

ES-MS, m/z 412.2 (M+1)⁺.

Analysis for C₂₃H₃₃N₅O₂·1.0 HCl·0.9H₂O: Calcd: C 59.51; H 7.77; N 15.09;Found: C 59.63; H 7.89; N 15.09.

Analytical HPLC (Method 2): >98%, t_(r)=11.3 min.

EXAMPLE 36

1-(5-Chloroindole-2-carbonyl-D-ethylglycinyl)-4-(1-methyl-piperidin-4-yl)piperazineHydrochloride.

Prepared from l-D-ethylglycinyl-4-(1-methylpiperidin-4-yl)piperazinetrihydrochloride and 5-chloroindole-2-carboxylic acid using methodssubstantially equivalent to General Coupling Method 1. The HCl salt isprepared following General Salt Formation Method 1.

¹H NMR.

ES-MS, m/z 446.2 (M+1)⁺.

Analysis for C₂₃H₃₂N₅O₂Cl·1.0 HCl·2.0H₂O: Calcd: C 53.28; H 7.19; N13.51; Cl 13.68; Found: C 53.14; H 6.51; N 13.29; Cl 13.45.

Analytical HPLC (Method 2): >99%, t_(r)=18.9 min.

EXAMPLE 37

1-(4-Methoxybenzoyl-D-ethylglycinyl)-4-(1-methylpiperidin-4-yl)piperazineHydrochloride.

Prepared from 1-D-ethylglycinyl-4-(1-methylpiperidin-4-yl)piperazinetrihydrochloride and 4-methoxybenzoyl chloride using methodssubstantially equivalent to General Coupling Method 4. The HCl salt isprepared following General Salt Formation Method 1.

¹H NMR.

ES-MS, m/z 403.3 (M+1)⁺.

Analysis for C₂₂H₃₄N₄O₃·HCl·0.4H₂O: Calcd: C 59.35; H 7.88; N 12.59; Cl7.96; Found: C 59.38; H 8.21; N 12.54; Cl 8.05.

Analytical HPLC (Method 2): >99%, t_(r)=7.2 min.

EXAMPLE 38

1-(6-Chlorobenzo[b]thiophene-2-carbonyl-D-ethylglycinyl)-4-(1-methylpiperidin-4-yl)piperazineHydrochloride.

Prepared from 1-D-ethylglycinyl-4-(1-methylpiperidin-4-yl)piperazinetrihydrochloride and 6-chlorobenzo[b]thiophene-2-carboxylic acid usingmethods substantially equivalent to General Coupling Method 1. The HClsalt is prepared following General Salt Formation Method 1.

¹H NMR.

ES-MS, m/z 463.2 (M+1)⁺.

Analysis for C₂₃H₃₁N₄O₂SCl·HCl·1.4H₂O: Calcd: C 52.65; H 6.68; N 10.68;Found: C 52.72; H 6.70; N 10.34.

Analytical HPLC (Method 2): >99%, t_(r)=17.8 min.

EXAMPLE 39

1-[Indole-6-carbonyl-D-propylglycinyl]-4-(1-methylpiperidin-4-yl)piperazineHydrochloride.

Prepared from 1-D-propylglycinyl-4-(1-methylpiperidin-4-yl)piperazinetrihydrochloride and indole-6-carboxylic acid using methodssubstantially equivalent to General Coupling Method 1. The HCl salt isprepared following Salt Formation Method 1.

¹H NMR.

ES-MS, m/z 426.3 (M+1)⁺.

Analysis for C₂₄H₃₅N₅O₂·HCl·1.9H₂O: Calcd: C 58.20; H 7.90; N 14.14; Cl7.16; Found: C 58.31; H 7.75; N 14.07; Cl 7.14.

Analytical HPLC (Method 2): >99%, t_(r)=14.4 min.

EXAMPLE 40

1-[5-Chloroindole-2-carbonyl-D-propylglycinyl]-4-(1-methylpiperidin-4-yl)piperazineHydrochloride.

Prepared from 1-D-propylglycinyl-4-(1-methylpiperidin-4-yl)piperazinetrihydrochloride and 5-chloroindole-2-carboxylic acid using methodssubstantially equivalent to General Coupling Method 1. The HCl salt isprepared following General Salt Formation Method 1.

¹H NMR.

ES-MS, m/z 460.2 (M+1)⁺.

Analysis for C₂₄H₃₄N₅O₂Cl·0.9 HCl·2.4H₂O: Calcd: C 53.77; H 7.46; N13.06; C_(1-12.57; Found: C) 53.84; H 6.70; N 12.96; Cl 12.40.

Analytical HPLC (Method 2): >99%, t_(r)=19.8 min.

EXAMPLE 41

1-[4-Methoxybenzoyl-D-propylglycinyl]-4-(1-methylpiperidin-4-yl)piperazineHydrochloride.

Prepared from 1-D-propylglycinyl-4-(1-methylpiperidin-4-yl)piperazinetrihydrochloride and 4-methoxybenzoyl chloride using methodssubstantially equivalent to General Coupling Method 4. The HCl salt isprepared following General Salt Formation Method 1.

¹H NMR.

ES-MS, m/z 417.3 (M+1)⁺.

Analysis for C₂₃H₃₆N₄O₃·HCl: Calcd: C 60.98; H 8.23; N 12.37; Cl 7.83;Found: C 60.99; H 8.33; N 12.35; Cl 7.75.

Analytical HPLC (Method 2): >99%, t_(r)=11.6 min.

EXAMPLE 42

1-[6-Chlorobenzo[b]thiophene-2-carbonyl-D-propylglycinyl]-4-(1-methylpiperidin-4-yl)piperazineHydrochloride.

Prepared from 1-D-propylglycinyl-4-(1-methylpiperidin-4-yl)piperazinetrihydrochloride and 6-chlorobenzo[b]thiophene-2-carboxylic acid usingmethods substantially equivalent to General Coupling Method 1 withreverse phase purification as described in General Coupling Method 4 togive the dihydrochloride salt.

¹H NMR.

ES-MS, m/z 477.3 (M+1)⁺.

Analysis for C₂₄H₃₃N₄O₂SCl·3.3 HCl·0.1H₂O: Calcd: C 48.11; H 6.14; N9.35; Found: C 47.81; H 5.74; N 8.96.

Analytical HPLC (Method 2): >96%, t_(r)=19.5 min.

EXAMPLE 43

1-[Indole-6-carbonyl-D-butylglycinyl]-4-(1-methylpiperidin-4-yl)piperazineHydrochloride.

Prepared from 1-D-butylglycinyl-4-(1-methylpiperidin-4-yl)piperazinetrihydrochloride and indole-6-carboxylic acid using methodssubstantially equivalent to General Coupling Method 1. The HCl salt isprepared following General Salt Formation Method 1.

¹H NMR.

ES-MS, m/z 440.3 (M+1)⁺.

Analysis for C₂₅H₃₇N₅O₂·HCl·2.3H₂O: Calcd: C 58.14; H 8.12; N 13.56; Cl6.86; Found: C 58.15; H 7.46; N 13.41; Cl 6.94.

Analytical HPLC (Method 2): >99%, t_(r)=16.8 min.

EXAMPLE 44

1-[5-Chloroindole-2-carbonyl-D-butylglycinyl]-4-(1-methylpiperidin-4-yl)piperazineHydrochloride.

Prepared from 1-D-butylglycinyl-4-(1-methylpiperidin-4-yl)piperazinetrihydrochloride and 5-chloroindole-2-carboxylic acid using methodssubstantially equivalent to General Coupling Method 1. The HCl salt isprepared following General Salt Formation Method 1.

¹H NMR.

ES-MS, m/z 474.2 (M+1)⁺.

Analysis for C₂₅H₃₆N₅O₂Cl·HCl·1.5H₂O: Calcd: C 55.86; H 7.50; N 13.03;Cl 13.19; Found: C 55.95; H 7.00; N 12.94; Cl 13.09.

Analytical HPLC (Method 2): >99%, t_(r)=21.6 min.

EXAMPLE 45

1-[4-Methoxybenzoyl-D-butylglycinyl]-4-(1-methylpiperidin-4-yl)piperazineHydrochloride.

Prepared from 1-D-butylglycinyl-4-(1-methylpiperidin-4-yl)piperazinetrihydrochloride and 4-methoxybenzoyl chloride using methodssubstantially equivalent to General Coupling Method 4. The HCl salt isprepared following General Salt Formation Method 1.

¹H NMR.

ES-MS, m/z 431.3 (M+1)⁺.

Analysis for C₂₄H₃₈N₄O₃·HCl·0.8H₂O: Calcd: C 60.00; H 8.31; N 11.66; Cl7.38; Found: C 60.00; H 8.35; N 11.54; Cl 7.30.

Analytical HPLC (Method 2): >99%, t_(r)=14.7 min.

EXAMPLE 46

1-[6-Chlorobenzo[b]thiophene-2-carbonyl-D-butylglycinyl]-4-(1-methylpiperidin-4-yl)piperazineHydrochloride.

Prepared from 1-D-butylglycinyl-4-(1-methylpiperidin-4-yl)piperazinetrihydrochloride and 6-chlorobenzo[b]thiophene-2-carboxylic acid usingmethods substantially equivalent to General Coupling Method 1. The HClsalt is prepared following General Salt Formation Method 1.

¹H NMR.

ES-MS, m/z 491.2 (M+1)⁺.

Analysis for C₂₅H₃₅N₄O₂SCl·HCl·2H₂O: Calcd: C 53.28; H 7.15; N 9.94;Found: C 52.92; H 6.84; N 10.23.

Analytical HPLC (Method 2): >99%, t_(r)=21.4 min.

EXAMPLE 47

1-(5-Chloroindole-2-carbonyl-D-propargylglycinyl)-4-(1-methylpiperidin-4-yl)piperazine.

Method D-1: To a mixture of1-(D-propargylglycinyl)-4-(1-methylpiperidin-4-yl)piperazine (725 mg,2.6 mmol), 5-chloroindole-2-carboxylic acid (510 mg, 2.6 mmol), HOBt(363 mg, 2.6 mmol), DMF (8 mL), and triethylamine (0.72 mL, 5.7 mmol) isadded DCC (700 mg, 3.4 mmol). The mixture is stirred overnight at roomtemperature. The precipitate that formed is removed by filtration andwashed with DMF. The filtrate is concentrated under vacuum. The residueis dissolved in methanol and applied to SCX resin (activated with 5%acetic acid and rinsed with methanol), washed with methanol and elutedwith saturated ammonia/methanol solution. The product fractions areconcentrated under vacuum, and the residue is purified by chromatographyon silica gel (dichloromethane and CMA) to provide the titled compound(730 mg, 62%).

¹H NMR (CDCl₃).

APCI-MS, m/e=456 (M+1).

EXAMPLE 48

1-(5-Chloroindole-2-carbonyl-D-propargylglycinyl)-4-(1-methylpiperidin-4-yl)piperazineHydrochloride.

Salt Formation Method 3: To a solution of1-(5-chloroindole-2-carbonyl-D-propargylglycinyl)-4-(1-methylpiperidin-4-yl)piperazine(700 mg, 1.54 mmol) in acetonitrile (200 mL) is added hydrogen chloride(1.54 mL, 1.54 mmol, 1 N in diethyl ether) dropwise at 0° C. The solventis evaporated under vacuum. The residue is dissolved in methanol,concentrated under vacuum, and dried in vacuum oven at 40° C. to providethe titled compound (768 mg, quantitative).

[α]²⁵ _(D) −23.0° (c 0.30, Methanol)

Melting Point=174-185° C. with decomposition.

¹H NMR (CD₃OD).

APCI-MS, m/e=456 (C₂₄H₃₀ClN₅O₂+1).

TLC R_(f)=0.34 (3:2 CH₂Cl₂:CMA)

Analysis for C₂₄H₃₀ClN₅O₂·0.9 HCl·0.6H₂O: Calcd: C, 57.38; H, 6.47; N,13.92; Cl, 13.76; Found: C, 57.47; H, 6.50; N, 13.76; Cl, 13.74.

HPLC Analysis (Method A): >99% t_(r)=12.9 min.

EXAMPLE 49

1-(Indole-6-carbonyl-D-propargylglycinyl)-4-(1-methylpiperidin-4-yl)piperazine.

Using methods substantially equivalent to that described in Method D-1,the subtitled compound is prepared from1-(D-propargylglycinyl)-4-(1-methylpiperidin-4-yl)piperazine andindole-6-carboxylic acid (76%).

¹H NMR (CDCl₃).

APCI-MS, m/e=422 (M+1).

EXAMPLE 50

1-(Indole-6-carbonyl-D-propargylglycinyl)-4-(1-methylpiperidin-4-yl)piperazineHydrochloride.

Using methods substantially equivalent to those described in SaltFormation Method 3, the titled compound is prepared from1-(indole-6-carbonyl-D-propargylglycinyl)-4-(1-methyl-piperidin-4-yl)piperazine(96%).

[α]²⁵ _(D) −14.1° (c 0.29, Methanol)

Melting Point=170-180° C. with decomposition.

¹H NMR (CD₃OD).

APCI-MS, m/e=422 (C₂₄H₃₁N₅O₂+1).

TLC R_(f)=0.23 (3:2 CH₂Cl₂:CMA)

Analysis for C₂₄H₃₁N₅O₂·1.2 HCl·1.2H₂O: Calcd: C, 59.20; H, 7.16; N,14.38; Cl, 8.74; Found: C, 59.41; H, 7.01; N, 14.14; Cl, 8.93.

HPLC Analysis (Method A): >99% t_(r)=9.9 min.

EXAMPLE 51

1-(6-Chlorobenzo[b]thiophene-2-carbonyl-D-propargylglycinyl)-4-(1-methylpiperidin-4-yl)piperazine.

Using methods substantially equivalent to that described in Method D-1,the titled compound is prepared from1-(D-propargylglycinyl)-4-(1-methylpiperidin-4-yl)piperazine and6-chlorobenzo[b]thiophene-2-carboxylic acid (76%).

¹H NMR (CDCl₃).

APCI-MS, m/e=472 (M+1).

EXAMPLE 52

1-(6-Chlorobenzo[b]thiophene-2-carbonyl-D-propargylglycinyl)-4-(1-methylpiperidin-4-yl)piperazineHydrochloride.

Using methods substantially equivalent to those described in SaltFormation Method 3, the subtitled compound is prepared from1-(6-chlorobenzo[b]thiophene-2-carbonyl-D-propargyl-glycinyl)-4-(1-methylpiperidin-4-yl)piperazine(96%).

[α]²⁵ _(D) −19.1°(c 0.295, Methanol)

Melting Point=115-135° C. with decomposition.

¹H NMR (CD₃OD).

APCI-MS, m/e=473 (C₂₄H₂₉ClN₄O₂S+1).

TLC R_(f)=0.53 (3:2 CH₂Cl₂:CMA)

Analysis for C₂₄H₂₉ClN₄O₂S·HCl·0.9H₂O: Calcd: C, 54.83; H, 6.01; N,10.66; Cl, 13.49; Found: C, 54.66; H, 6.06; N, 10.54; Cl, 13.75.

HPLC Analysis (Method A): >99% t_(r)=13.2 min.

EXAMPLE 53

1-(Indole-6-carbonyl-D-allylglycinyl)-4-(1-methylpiperidin-4-yl)piperazine.

Using methods substantially equivalent to those described in Method D-1,the titled compound is prepared from1-(D-allylglycinyl)-4-(1-methylpiperidin-4-yl)piperazine andindole-6-carboxylic acid (62%).

¹H NMR (CDCl₃).

APCI-MS, m/e 424 (C₂₄H₃₃N₅O₂+1).

Example 54

1-(Indole-6-carbonyl-D-allylglycinyl)-4-(1-methylpiperidin-4-yl)piperazineHydrochloride.

Using methods substantially equivalent to those described in SaltFormation Method 3, the titled compound is prepared from1-(indole-6-carbonyl-D-allylglycinyl)-4-(1-methylpiperidin-4-yl)piperazine(86%).

[α]²⁵ _(D) −58.7° (c 0.25, Methanol)

Melting Point=110-115° C. with decomposition.

¹H NMR (CD₃OD).

APCI-MS, m/e 424 (C₂₄H₃₃N₅O₂+1).

TLC R_(f)=0.58 (3:7 CH₂Cl₂:CMA).

Analysis for C₂₄H₃₃N₅O₂·1.2 HCl·1.5H₂O: Calcd: C, 58.31; H, 7.58; N,14.17; Cl, 8.61; Found: C, 58.35; H, 7.67; N, 14.07; Cl, 8.33.

HPLC Analysis (Method A): >99% t_(r)=10.9 min.

EXAMPLE 55

1-(6-Chlorobenzo[b]thiophene-2-carbonyl-D-allylglycinyl)-4-(1-methylpiperidin-4-yl)piperazine.

Using methods substantially equivalent to those described in Method D-1,the subtitled compound is prepared from1-(D-allylglycinyl)-4-(1-methylpiperidin-4-yl)piperazine and6-chlorobenzo[b]thiophene-2-carboxylic acid (46%).

¹H NMR (CDCl₃).

APCI-MS, m/e 475 (C₂₄H₃₁ClN₄O₂S+1).

EXAMPLE 56

1-(6-Chlorobenzo[b]thiophene-2-carbonyl-D-allylglycinyl)-4-(1-methylpiperidin-4-yl)piperazineHydrochloride.

Using methods substantially equivalent to those described in SaltFormation Method 3, the titled compound is prepared from1-(6-chlorobenzo[b]thiophene-2-carbonyl-D-allylglycinyl)-4-(1-methylpiperidin-4-yl)piperazine(84%).

[α]²⁵ _(D) −48.5° (c 0.25, Methanol).

Melting Point=130-135° C. with decomposition.

¹H NMR (CD₃OD).

APCI-MS, m/e 475 (C₂₄H₃₁ClN₄O₂S+1).

TLC R_(f)=0.67 (3:7 CH₂Cl₂:CMA).

Analysis for C₂₄H₃₁ClN₄O₂S·0.9 HCl·0.9H₂O: Calcd: C, 55.00; H, 6.48; N,10.69; Cl, 12.85; S, 6.12; Found: C, 55.00; H, 6.46; N, 10.46; Cl,12.74; S, 5.88.

HPLC Analysis (Method A): >99% t_(r)=13.6 min.

EXAMPLE 57

1-(5-Chloroindole-2-carbonyl-D-allylglycinyl)-4-(1-methyl-piperidin-4-yl)piperazine.

Using methods substantially equivalent to those described in Method D-1,the titled compound is prepared from1-(D-allylglycinyl)-4-(1-methylpiperidin-4-yl)piperazine and5-chloroindole-2-carboxylic acid (73%).

¹H NMR (CDCl₃).

APCI-MS, m/e 458 (C₂₄H₃₂ClN₅O₂+1).

EXAMPLE 58

1-(5-Chloroindole-2-carbonyl-D-allylglycinyl)-4-(1-methyl-piperidin-4-yl)piperazineHydrochloride.

Using methods substantially equivalent to those described in SaltFormation Method 3, the subtitled compound is prepared from1-(5-chloroindole-2-carbonyl-D-allylglycinyl)-4-(1-methyl-piperidin-4-yl)piperazine(82%).

[α]²⁵ _(D) −60.1° (c 0.25, Methanol)

Melting Point=170-175° C. with decomposition.

¹H NMR (CD₃OD).

APCI-MS, m/e 458 (C₂₄H₃₂ClN₅02+1).

TLC R_(f)=0.57 (3:7 CH₂Cl₂:CMA).

Analysis for C₂₄H₃₂ClN₅O₂·1.1 HCl·0.8H₂O: Calcd: C, 56.24; H, 6.82; N,13.66; Cl, 14.53; Found: C, 56.33; H, 7.01; N, 13.43; Cl, 14.49.

HPLC Analysis (Method A): 99% t_(r)=13.1 min.

Enzyme Inhibition assays:

The ability of a test compound to inhibit factor Xa may be evaluated inone or more of the following Enzyme Inhibition assays, or in otherstandard assays known to those skilled in the art.

Enzyme Inhibition Assay

Human factor Xa and human thrombin are purchased from Enzyme ResearchLaboratories (South Bend, Ind., USA). Other proteases are from othercommercial sources. Chromogenic para-nitroanilide peptide proteasesubstrates are purchased from Midwest Biotech (Fishers, Ind., USA).

The binding affinities for human factor Xa are were measured as apparentassociation constants (Kass) derived from protease inhibition kineticsas described previously.^(a,b,c,d) The apparent Kass values are obtainedusing automated (BioMek-1000) dilutions of inhibitors (Kassdeterminations are performed in triplicate at each of four-eightinhibitor concentrations) into 96-well plates and chromogenic substratehydrolysis rates determined at 405 nm using a Thermomax plate readerfrom Molecular Devices (San Francisco). For factor Xa inhibition, theassay protocol is: 50 μL buffer (0.06 M tris, 0.3 M NaCl, pH 7.4); 25 μLinhibitor test solution (in MeOH); 25 μL human factor Xa (32 nM in 0.03M tris, 0.15 M NaCl, 1 mg/mL HSA); finally, 150 μL BzIleGluGlyArgpNA(0.3 mM in water) added within 2 min to start hydrolysis. Final [factorXa] is 3.2 nM. [Free Xa] and [bound Xa] are determined from linearstandard curves on the same plate by use of SoftmaxPro software for eachinhibitor concentration and apparent Kass calculated for each inhibitorconcentration which produced hydrolysis inhibition between 20% and 80%of the control (3.2 nM factor Xa): apparent Kass[E:I]/[E_(f)][I_(f)]=[E_(b)]/[E_(f)][I^(o)−I_(b)]. The apparent Kassvalues so obtained are approximately the inverse of the Ki for therespective inhibitors [1/appKass=app Ki]. The variability of meanapparent Kass values determined at the single substrate concentration is+/−15%. The assay system Km was measured as 0.347+/−0.031 mM [n=4]; andVmax was 13.11+/−0.76 μM/min.

Kass values are determined with thrombin and other proteases using thesame protocol with the following enzyme and substrate concentrations:

-   thrombin, 5.9 nM with 0.2 mM BzPheValArgpNA;-   factor XIa, 1.2 nM with 0.4 mM pyroGluProArgpNA;-   factor XIIa, 10 nM with 0.2 mM HDProPheArgpNA;-   plasmin, 3.4 nM with 0.5 mM HDValLeuLyspNA;-   nt-PA, 1.2 nM with 0.8 mM HDIleProArgpNA;-   urokinase, 0.4 nM with 0.4 mM pyroGluGlyArgpNA;-   aPC, 3 nM with 0.174 mM pyroGluProArgpNA;-   plasma kallikrein, 1.9 nM with D-ProPheArgpNA; and-   bovine trypsin, 1.4 nM with 0.18 mM BzPheValArgpNA.

Citations

-   (a) Sall D J, J A Bastian, S L Briggs, J A Buben, N Y Chirgadze, D K    Clawson, M L Denny, D D Giera, D S Gifford-Moore, R W Harper, K L    Hauser, V J Klimkowski, T J Kohn, H-S Lin, J R McCowan, A D    Palkowitz, G F Smith, M E Richett, K Takeuchi, K J Thrasher, J M    Tinsley, B G Utterback, S-CB Yan, M Zhang. Dibasic    Benzo[b]thiophenes Derivatives as a Novel Class of Active Site    Directed Thrombin Inhibitors. 1. Determination of the Serine    Protease Selectivity, Structure-Activity Relationships and Binding    Orientation. J Med Chem 40 3489-3493 (1997).-   (b) Smith G F, T J Craft, D S Gifford-Moore, W J Coffman, K D Kurz,    E Roberts, R T Shuman, G E Sandusky, N D Jones, N Chirgadze, and C V    Jackson. A Family of Arginal Thrombin Inhibitors Related to    Efegatran. Sem. Thrombos. Hemost. 22, 173-183 (1996).-   (c) Smith G F, D S Gifford-Moore, T J Craft, N Chirgadze, K J    Ruterbories, T D Lindstrom, J H Satterwhite. Efegatran: A New    Cardiovascular Anticoagulant. In New Anticoagulants for the    Cardiovascular Patient. Ed. R Pifarre. Hanley & Belfus, Inc.,    Philadelphia (1997) pp 265-300.-   (d) Sall D J, D L Bailey, J A Bastian, N Y Chirgadze, A C    Clemens-Smith, M L Denney, M J Fisher, D D Geira, D S Gifford-Moore,    R W Harper, L M Johnson, V J Klimkowski, T J Kohn, H S Lin, J R    McCowan, A D Palkowitz, M E Richett, G F Smith, D W Snyder, K    Takeuchi, J E Toth, M Zang. Diamino Benzo[b]thiophene Derivatives as    a Novel Class of Active Site Directed Thrombin Inhibitors: 5.    Potency, Efficacy and Pharmacokinetic Properties of Modified C-3    Side Chain Derivatives. J. Med. Chem., 43, 649-663 (2000).

The compounds of formula (I) exemplified herein have been found toexhibit a Kass of greater than 1×10⁶ L/mole in the enzyme inhibitionassay. For example, the compounds, or their pharmaceutically acceptablesalts, listed hereinabove as preferred and exemplified in Examples 6, 9and 18 have been to exhibit Kass values of about 20, 89 and 358×10⁶L/mole, respectively.

The ability of a test compound to elongate Partial Thromboplastin Time(Prothrombin Time) may be evaluated in the following test protocols.

Partial Thromboplastin Time (Prothrombin) Test Protocol

Venous blood is collected into 3.2% (0.109 M) trisodium citratevacutainer tubes at 1 volume of anticoagulant to nine volumes of blood.The blood cells are separated by centrifugation at 700 g for ten minutesto yield plasma, which is frozen at 70° C. until required.

To perform the test, 100 μL of plasma are pipetted into in a glass testtube, 1 μL of test compound in DMSO is added, and allowed to warm to 37°over two minutes. 100 μL of warm (37°) Manchester (tissue thromboplasin)reagent (Helena Biosciences, UK) is added, allowed to equilibrate fortwo minutes. 100 μL of warm (37° C.) 25 mM calcium chloride solution isadded to initiate clotting. The test tube is tilted three times througha 900 angle every five seconds to mix the reagents and the time to clotformation recorded. Data from a series of observations and test compoundconcentrations are analysed by a SAS statistical analysis program and aCT2 (Concentration required to double clotting time) for each compoundis generated.

The compound of the invention has been found to significantly elongatethe partial thromboplastin time (Prothrombin time).

Alternative Prothrombin Time and APTT Protocols

Coagulation Determinations: Prothrombin Times and APTT values aredetermined in HUMAN PLASMA with a STA instrument (Stago). BioPT is aspecial non-plasma clotting assay triggered with human tissue factor(Innovin). Possible binding to albumen or to lipid are assessed bycomparing the BioPT effects in the presence/absence of 30 mg/mL humanalbumen (HSA) and 1 mg/mL phosphatidyl choline (PC). Inhibitors aredelivered in 50% aqueous methanol vehicle.

APTT ASSAY

-   75 μL plasma Citrol Baxter-Dade Citrated Normal Human Plasma-   25 μL test solution-   75 μL Actin Baxter-Dade Activated Cephaloplastin incubate 2 min-   min. @ 37° C.-   75 μl CaCl₂ (0.02 M)    PT ASSAY-   75 μL plasma-   25 μL test solution-   75 μL saline incubate 1 min. @ 37° C.-   75 μL Innovin Baxter-Dade Recombinant Human Tissue Factor

1. A compound of formula (I)

in which R¹ represents (1-4C)alkyl, (2-4C)alkenyl or (2-4C)alkynyl; andR² is selected from

in which X¹ represents a hydrogen atom or a halogen atom; X² representsa hydrogen atom, a methyl group, a chlorine atom or a bromine atom; X³represents a hydrogen atom, a methyl group or a halogen atom; X⁵represents chloro, methoxy or methyl; X⁶ represents a hydrogen atom, ahalogen atom or a methyl group; and X⁴ represents CH or N; or apharmaceutically acceptable salt thereof.
 2. A compound as claimed inclaim 1, in which R² is selected from


3. A compound as claimed in claim 2, in which R² is selected from


4. A compound as claimed in claim 1, in which R² is selected from


5. A compound as claimed in claim 1, in which R¹ is a (1-4C)alkyl group.6. A compound as claimed in claim 3, in which R¹ is methyl, ethyl,propyl, 2-propyl, butyl, t-butyl, 1-methylpropyl or 2-methylpropyl.
 7. Acompound as claimed in claim 6, in which R¹ is 2-propyl.
 8. A compoundas claimed in claim 1, in which R¹ is methyl, ethyl, propyl, 2-propyl,butyl, t-butyl, 1-methylpropyl, 2-methylpropyl, prop-2-enyl orprop-2-ynyl.
 9. A compound as claimed in claim 1, in which X¹ representsa hydrogen atom or a fluorine atom; X² represents a hydrogen atom or achlorine atom; X³ represents a fluorine or chlorine atom; and X⁶represents a chlorine atom.
 10. A compound as claimed in claim 9, inwhich R² is 4-methoxyphenyl, indol-6-yl, 3-methylindol-6-yl,3-chloroindol-6-yl, 5-chloroindol-2-yl, 3-fluoro-4-methoxyphenyl,5-fluoroindol-2-yl or 6-chlorobenzo[b]thiophen-2-yl.
 11. A compound asclaimed in claim 3, in which X¹ represents a hydrogen atom or a fluorineatom; X² represents a hydrogen atom or a chlorine atom; and X³represents a chlorine atom.
 12. A compound as claimed in claim 11, inwhich R² is 4-methoxyphenyl, indol-6-yl, 3-methylindol-6-yl,3-chloroindol-6-yl, or 5-chloroindol-2-yl.
 13. A compound as claimed inclaim 11, in which R² is 4-methoxyphenyl, indol-6-yl or5-chloroindol-2-yl.
 14. A compound as claimed in claim 1, in whichX⁴represents CH.
 15. A compound as claimed in claim 1, which is selectedfrom:1-(4-methoxybenzoyl-D-valinyl)-4-(1-methylpiperidin-4-yl)piperidine and1-(indole-6-carbonyl-D-valinyl)-4-(1-methylpiperidin-4-yl)piperidine;and pharmaceutically acceptable salts thereof.
 16. A compound as claimedin claim 1, in which X⁴ represents N.
 17. A compound as claimed in claim1, which is selected from:1-(5-chloroindole-2-carbonyl-D-valinyl)-4-(1-methyl-piperidin-4-yl)piperazine,and pharmaceutically acceptable salts thereof.
 18. A pharmaceuticalcomposition, which comprises a compound as claimed in claim 1, togetherwith a pharmaceutically acceptable diluent or carrier.
 19. A process forpreparing a compound as claimed in claim 1, which comprises (a) reactinga compound of formula (II)

or a salt thereof, with a compound of formula (III)

or a reactive derivative thereof; or (b) reacting a compound of formula(IV)

or a salt thereof, with a compound of formula (V)HOOC—R²  (V) or a reactive derivative thereof; followed, if apharmaceutically acceptable salt is desired, by forming apharmaceutically acceptable salt.
 20. A compound of formula (III)

or a salt thereof, in which R¹ and R² are as defined in claim
 19. 21. Acompound of formula (IV)

or a salt thereof, in which R¹ and X⁴ are as defined in claim
 19. 22.(canceled):
 23. (canceled):
 24. A method of treating a thromboticdisorder in a subject requiring treatment, which comprises administeringan effective amount of a compound as claimed in claim 1.